Team:Lethbridge/Notebook

From 2009.igem.org

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Latest revision as of 20:11, 21 October 2009

Calendar

May

May 5

Roxanne, Megan, Alix, Mackenzie, Sebastian

prepared Lb Media in test tubes -prepared 250mL LB agar for plates

Prepared 500nM Tris-HCl (pH 8.0), EDTA (pH 8.0, 500nm) TE Buffer (pH 8.0), GTE (Solution I), 2M NaOH, 10% SDS

May 6

Roxanne

Autoclaved LB test tubes, LB agar, Tris-HCl, EDTA, TE, GTE

Added ampicillin to LB agar (100ug/mL) -Poured LB + amp plates

May 12

Roxanne

Prepared LB agar
added Tetracyclin (100ug/mL) –
Poured LB + ampt plates

Roxanne, Alix, Ashley, Megan, Mackenzie, Kirsten

transferred pE43 (DH5α) cells from gylcerol stock to 5mL LB+Tet test tube (x8)
placed in orbital shaker overnight at 37°C

May 13

Roxanne

Removed test tubes from shaker (16hrs incubation) and placed in deli fridge

May 14

Roxanne, Alix, Ashley, Mackenzie, Kirsten

Preformed alkaline lysis miniprep on pE43 plasmid -lost plasmid from 2 tubes

obtained 6X pE43 plasmid (2X ohbR, 2x ohbB, ohbA, ohbC)

May 19

Roxanne, Alix, Ashley, Kirsten, Megan, Mackenzie

Gel Electrophoresis : 1% agarose in TAE

Lane 1: Ladder

Lane 2: ohbR1

Lane 3: ohbR2

Lane 4: ohbC

Lane 5: ohbB1

Lane 6: ohbB2

Lane 7:ohbA

Lane 8:ohbC

Lane 9: ohbB1

Lane 10:ohbB2

DNA quantification by UV spec 260nm : 2.013 280nm: 1.105 260/280 : 1.984 Concentration: 2517µg/mL PCR of ohbA, (ohbB)x2, ohbC, (ohbR)x2

Took picture of the gel - it did not turn out.

May 21

Roxanne

Made 50X TAE Buffer
Running PCR amplicons & RS/pSB1A2 ligation product on a 1% agarose gel
Jeff gave tutorial on primer design and quick-change mutagenesis to Kirsten, Ashley, Mackenzie, Fan, Lisza, Roxanne

May 26

Roxanne, Alix, Megan, Mackenzie, Kirsten

Gel did not turn out gave tutorial on open wetware and the igem registry

May 28

Roxanne

reran the gel from may 19th, only primer dimers present

Roxanne, Ashley, Alix, Kirsten, Mackenzie, Megan

Redoing the PCR of ohbA, ohbB, ohbC and OhbR with Phusion DNA Polymerase

Roxanne

refilled the tip boxes

May 29

Roxanne

prepared glycerol stocks for autoclaving

June

June 1

Roxanne

Ran a 1% agarose in TAE gel of the PCR products from May 28th.
5µL of ladder, 2µL of DNA

Lane 1: ladder

Lane 2: ohbA (conc.1x)

Lane 3: ohbA (conc.1/10)

Lane 4: ohbB(1x)

Lane 5: ohbB(1/10)

Lane 6: ohbC(1x)

Lane 7: ohbC(1/10)

Lane 8: ohbR (1x)

Lane 9: ohbR(1/10)

Lane 10: ohbR(1x)

Gel did not show any amplification

Mackenzie & Roxanne

Transformation of pSB1A3 into DH5α

Made 2 aliquots of pSB1A3 and 1 of pUC19

plated pSB1A3-1: straight and dilute, pSB1A3-2: straight and dilute, pUC19: straight only incubated overnight at 37°C

June 3

Roxanne

positive control worked
Think the transformation worked but slight problem. The plasmid contains a suicide gene. Therefore, any cells that took up the plasmid would die.
planning new genes to transform
GENE LOCATION pSB1A3 + IPTG Inducible RFP 1-1k Bba_I13522, pTET GFP 2-8A Bba_I13521, pTet mRFP 2-6O Bba_I13600, pTET CFP 1-24E BBA-B0015, double terminator 1-23L

June 4

Picked some glycerol stock GFP for the AIF visit

made ampicillin plates 371.39g/mol X100mmol/LX0.002L = 74.278mg

June 15

Transformed the pTet and EYFP genes from the Biobrick registry

June 16

Picked colonies in the early morning for Mini prep in the afternoon

Restriction digested the plasmids: pTet with SpeI and PstI, EYFP with XbaI and PstI

June 17

Quenched the restriction digests from the day before

June 18

Checked the concentrations of:
- Riboswitch-1 : 4549u/.mL
- Riboswitch-2 : 4283ug/mL
- rpsA TIR-1 : 106ug/mL
- rpsA TIR-2 : 90ug/mL
- rpsA TIR-3 : 78ug/mL
- rpsA TIR-4 : 74ug/mL
- rpsA TIR-4b : 67ug/mL
- rpsA TIR-5 : 70ug/mL

in order to send for sequencing with the UR and UF2 primers

June 22

Picked one colony each from DH5a + pTet and DH5a + EYFP.
Grown overnight at 37 degrees in LB +100ug/mL Amp (5mL culture tubes)
From mini-preps of EYFP(1&2) and pTet (1&2) did preparative restriction digests
- pTet with PstI and SpeI
- EYFP with XbaI and PstI
In 37 degree water bath for 1hr and 20 min. Quenched at 60 degrees for 15 minutes.

June 23

Ran a 1% agarose gel (1mL 50X TAE with 49mL Milli-Q H2O and 0.5g of agarose).

Lane 1: 1kb ladder

Lane 2:EYFP1A

Lane 3: EYFP1B

Lane 4: EYFP1B runover

Lane 5: EYFP2A

Lane 6: EYFP2B

Lane 7: pTet-1A

Lane 8: pTet-1B

Lane 9: pTet2-A

Lane 10: pTet-2B

Samples were 2uL of dye and 10uL of DNA.
The gel was unsuccessful. Possible reasons include: too short of an incubation during restriction digest or the enzymes were not functioning properly

June 24

Mini-Preps of pTet and EYFP
- 750uL of culture from the fridge into microcentrifuge tubes. Pellet at 13000rpm for 2 min. Removed supernatant and added the rest of the culture. Repelleted at 13000rpm for 2 min.
- followed protocol according to Qiagen mini-prep protocol on page 2
Restriction Digest of EYFP and pTet
- EYFP restricted with XbaI and PstI
- pTet restricted with PstI and SpeI
  - Double digest:1uL buffer tango, 8uL DNA, 0.5uL of each enzyme
  - Single digest (1 for each enzyme): 0.5uL water, 1uLbuffer tango, 8uL DNA, 0.5uL enzyme
  - Negative control: 1uL water, 1uL buffer, 8uL DNA
  - 8 tubes total in to the thermocycler for 8hrs at 37, 15min at 60 then held at 4 before going into the -20 fridge.

June 25

Ran a gel of the digested EYFP and pTet and controls from the previous day.
Used 0.3g of agarose in 30mL of 1X TAE
made stock solution of 1x TAE
Ran gel at 100V for 1hr

Lane 1: 1kb ladder

Lane 2: Control-no enzyme(pTet)

Lane 3: pTet-pstI

Lane 4: pTet-SpeI

Lane 5: pTet-SpeI/PstI

Lane 6:EYFP-no enzyme

Lane 7: EYFP-PstI

Lane 8: EYFP-XbaI

Lane 9: EYFP-XbaI/PstI

Lane 10: 1kb ladder

Gel melted
Made 2 pTet and 2EYFP 5mL culture tubes with LB and Amp(100mg/mL). Left in incubator overnight at 37°C

June 26

Cells harvested from pTet and EYFP cultures into 4 tubes. Spun down and LB supernatant removed. Stored in -20 for future use.

June 30

200mL cultures of cells with pTet, EYFP and CFP grown in LB and 100ug/mL Amp harvested by centrifugation then maxi-prepped.
Maxipreps
- incubated cell pellets in 3mL of ALSI and incubated at RT for 15min
- 6mL of ASL2 added and incubated at RT for 10min
- 4.5mL of AlS3 added and placed on ice for 10min
- Spun at 5000g at 4 for 15min
- 5mL of each phenol and chloroform added to tubes containing resulting supernatant
- spun for 10min at 4 and 5000g, upper aqueous layer saved
- 5mL of chloroform added to aqueous layer and spun for 10 min at 4 and 5000g. Supernatant saved
- 0.6 volumes isopropanol added to each tube and placed in -20 freezer for 30min –
- spun for 10min at 4 and 5000g
- pellet wasted with EtOH and airdried overnight

5 culture (500mL) flasks of LB made: 10g tryptone, 5g yeast extract, 10g salt and 1L water

July

July 2

DNA samples dissolved in 1000uL water, 100uL of RNAse added. Made 1/10, 1/100 and 1/1000 dilutions of pTet, CFP and EYFP.

July 3

Ran a 1% agarose gel at 120V for 60 minutes.
2uL DNA samples with 2uL 6x loading dye

Lane 2: 10uL of 2kb ladder,

Lane 4:undiluted pTet

Lane 5: undiluted CFP

Lane 6:undiluted EYFP

Lane 8: 1/10pTet

Lane 9: 1/10 CFP

Lane 10: 1/10 EYFP

Lane 12: 1/100 pTet

Lane 13: 1/100 CFP

Lane 14: 1/100 EYFP

Lane 16: 1/1000pTet

Lane 17: 1/1000 CFP

Lane 18: 1/1000 EYFP

July 6th

Jeff, Mackenzie, Ashley

Using 1/10 and 1/100 dilutions of pTET and EYFP to perform large scale digests.

pTET:PstI and SpeI
EYFP:XbaI and PstI

4 reactions each:

single digest (PstI/SpeI and XbaI/PstI)
double digest
no digest

100µL each, means we need 400µL total for each.

1/10 dilution=40µL DNA, 360µL water

1/100 dilution=4µL DNA, 396µL water

Reaction set up:

88µL DNA
10µL buffer Tango (10x)
1µL RE1 (or water)
1µL RE2 (or water)
=100 µL total

16 reactions run overnight @ 37° C

Kirsten, Lisza

TetR Q04400-plate 1 well 16p-pSB2K3

LacI promoter-R0010-plate 1 well 1d-Amp

pBAD-I13453-plate 1, well 1n-pSB1A2

strong RBS-B0030-plate 1 well 1h-pSB1A2

med RBS-B0030-plate 1 well 2I-pSB1A2

10µL water into wells

Transformation:

2µL DNA (TetR, LacI, pBAD, med RBS, strong RBS) into 25µL competent DH5α cells (from the -80 fridge). Pipet up then down and swirl. One ice for 30min, then heat shock at 42° C for 45 sec. Ice for 1 min, add 250µL LB broth (from fridge) and incubate in shaker for 1 hour. Plate onto ampicillin plates (pBAD, LacI, strond and med RBS) and kanamycin plate (TetR) into incubator overnight. Control: 2.5µL water and 2.5µL DH5α.

July 8

Jeff

Overnight cultures (200mL + antibiotics) of:

PlacI (Amp)
TetR (Kan)
Med RBS (Amp)

Centrifuged at 5000g, 10 min, 4° for maxiprep

500µL each of each culture used to make glycerol stocks – duplicates made, stored in -80° freezer after flash freezing with liquid nitrogen.

EYFP insert from AGE separation (incubated in 600µL buffer QG overnight):

+200µL isopropanol
Incubated on a QiaQuick column
13000rpm, 1 min

Flow through replaced in original tube. 500µL QG applied to column, 1 min centrifugation. Colum washed w/ 750µL PE buffer (1 min centrifuge). Flow through discarded. 1 more min of centrifugation to get rid of buffer PE. DNA eluted w/ 25µL water (37°C for 10 min) also a second water elution w/ 25µL water (E2).

1/10 double digest of pTet heat incubated at 85°C for 10 min.

Maxipreps: 3mL ALSI added, cells responded. 500µL lysozyme added. 15 min @RT, 6mL ALSII added. 10 min at RT. 4.5mL cold ALSIII added, 10 min on ice. Left a 4°C

July 13

Setting up ligations:

1µL ligase (stock)

10x T4 DNA ligase buffer

1/10 double digested pTet est 20ng/µL

E1 (60% yield) = ~5ng/µL; ~1/3 insert size/pTet

Control needed:

1/10 double digested pTet, no ligase
1/10 double digested pTet + ligase
1/10 double digested pTet + 3x vol of insert
1/10 double digested pTet +6x vol of insert

Reaction 1:

1µL T4 DNA ligase 10x buffer
1µL double digest pTet
8µL water

Reaction 2:

1µL T4 DNA ligase 10x buffer
1µL double digest pTet
0.5 µL T4 ligase
7.5µL water

Reaction 3:

1µL T4 DNA ligase 10x buffer
1µL double digest pTet
0.5 µL T4 ligase
3µL E1 EYFP insert
4.5µL water

Reaction 4:

1µL T4 DNA ligase 10x buffer
1µL double digest pTet
0.5 µL T4 ligase
6 µL E1 EYFP insert
1.5µL water

Incubated at 37°C overnight

Kirsten, Mackenzie Chloroform extraction of medRBS, pLacI, TetR, that Jeff started. pBab & strong RBS cloudy and phenol chloro done twice.

July 14

Megan, Mackenzie, Alix

Transformations of ligation reactions

Took 2µL of DNA from 1, 2, 3, 4 and added to 25 µL of DH5α cells. Incubated on ice for 30 min. Heat shocked at 42°C for 45 sec. Back to ice for 1 min. For ligations add 500µL of LB broth to each tube, working sterile using a Bunsen burner. Incubate in shaker at 37°C for1 hour at 200 rpm. Made up two plates (Amp) of each, 100µL and 400µL.

July 16th

Ashley, Mackenzie, Roxanne

Pelleted pTet-EYFP ligation reactions 3 and 4 using at 13000rpm for 1 min.

Mini-prep of pTet-EYFP ligation reactions 3 and 4 using Qiagen kit + protocol.

July 20th

Lisza

Our genes: added miiliQ water

10µL to N-term Arg tail, concentration = 200ng/µL
10µL to c-term Arg tail, concentration= 200ng/µL
20 µL to lumazine, concentration= 250ng/µL

July 21st

Lisza

Made glycerol stocks of the 4 pTet-EYFP ligations

Kirsten, Alix

Mini-prep of pTet-EYFP (3 &4)x2 followed protocol according to Qiagen.

Analytic restriction digest of above mini-preps

Control used mini prepped pTet from June 24.

Added (in order) to 1.5mL micro centrifuge tubes:

12µL water
2 µL buffer tango
5 µL DNA
0.5µL XbaI
Total: 20 µL

Megan, Kirsten, Alix, Lisza

Made an 80mL agarose gel for large rig

0.8g agarose

80mL 1x TAE

Lanes

Ladder 1Kb
Blank
pTet unrestricted
pTet undigested
rxn III-1 undigested (pTet-EYFP)
rxn III-1 digest
rxn III-2 undigested
rxn III-2 digest (XbaI)
blank
rxn IV-1 undigested
rxn IV-1 digest (XbaI)
rxn IV-2 undigested
rxn IV-2 digest (XbaI

Ran at 120V for 40 min( in @ 5:45) stained in ethidium bromide for 10 20 min.

Picture:

Lisza

Made glycerol stocks of maxiprep cultures:

3 lumazine

2 Arg N-term

1 Arg c-term

Spun down cultures

Lumazine-1=1.35g

Lumzine-2= 1.31g

Lumazine-3=1.29g

Arg c-term=1.26g

Arg n-term-1=1.43g

Arg n-term-2=1.12g

July 23

Did a colony screen PCR:

24 colonies from July 14th DH5α Amp

AB+MC+MT plates labeled 10-32

With picked colonies transferred bacteria into a 1.5mL tube with 300µL milliQ water. With same toothpick inoculated 5mL (Amp 5mL) grown overnight at 37°C. Put 1.5L tubes onto heat block at 95°C for 7 min. Centrifuged at full speed for 1 min. Used 5µL supernatant for PCR survey.

Reactions: control with EYFP

10.4µL water (14.9 µL control)
2 µL Taq buffer
0.4 µL dNTPs
1 µL VR primer
1 µL VFZ primer
0.2 µL Taq polymerase
5 µL template (0.5 µL control)

Cycle: Colony58

Note: tube 11 contained both samples 11 and 12

July 24th

1% agarose gel at 100V of colony PCR

Lanes:

1 kb Ladder (8 µL)
Control
Colony 10 (10 µL sample+2 µL dye)
Colony 11/12
Colony 13
Colony 14
Colony 15
Colony 16
Colony 17
Colony 18
Colony 19
Colony 20
Colony 21
Colony 22
Colony 23
Colony 24
Colony 25
Colony 26
Colony 27
Colony 28
Colony 29
Colony 30
Colony 31
Colony 32
Colony 33
1 Kb ladder (8 µL)

PCR was successful, but had negative colonies. No pTet EYFP biobrick, only pTet was in the plasmid

July 27

Fan & Lisza

Preparative restriction digest of pTet-EYFP:

50µL reactions
40 µL DNA
5 µLbuffer
0.5 µL RE1
0.5 µL RE@
4 µL milliQ water

pTet:

RE1=pstI
RE2=speI

EYFP:

RE1=pstI
RE2=xbaI

Incubated at 37°C for 2 hours

Kirsten, Lisza, Mackenzie

Purified double digested pTet using QiaQuick PCR purification kit. First run through eluted DNA with 50 µL water then done again and eluted with 30 µL elution buffer. Into -20°C fridge

Gel Extraction of EYFP

Weight of tube: 1.0973g

Weight of tube+gel: 1.9759g

Weight gel: 0.8786

Transferred to falcon tube

Preformed gel extraction according to Qiaquick Gel Extraction kit. Eluted with 30 µL of elution buffer.

July 28

Kirsten, Alix, Lisza

Ligation of pTet and EYFP

Reaction 1

1.55µL of water
2.5 µL 10x buffer
5 µL vector (pTet)
14.7 µL insert (EYFP)
1.25 µLT4 DNA ligase
25 µL total

Reaction 2 (Control)

16.25µL of water
2.5 µL 10x buffer
5 µL vector (pTet)
0.0µL insert (EYFP)
1.25 µLT4 DNA ligase
25 µL total

Incubate at room temperature for 2 hours then transform

Restriction Digest of Lumazine 1&2, C-terminal Arg tag, N-terminal Arg tag 1&2.

14.6µL of water
2.5 µL 10x buffer tango
4 µL DNA
0.4µ EcoRI
21 µL total

Incubate at 37°C for 2 hours in water bath.

July 28

Fan

Did a transformation with ligation reactions plus a water control.

July 29

Running gel of the PCR for EYFP and CFP with c-terminal fusion tags and the restriction digests of n-terminal, c-terminal ARG tags and the lumazine gene.

Loading 6µL ladder, 5µL sample+1µL dye

Lane:

1kb DNA ladder
CFP amplicon
EYFP amplicon
Empty
N-terminal #1 (EcoRI digestion)
N-terminal #2 (EcoRI digestion)
C-terminal (EcoRI digestion)
Lumazine # 1 (EcoRI digestion)
Lumazine # 2 (EcoRI digestion)

Did colony PCR of the 5 colonies from the EYFP-1 plater, 5 of the EYFP-2 plate, 4 of the pTet-1, 4 of the pTet-2, one negative control and one positive (EYFP plasmid). Followed protocol of July 23.

Megan, Lisza, McKenzie

Purify maxipreps using kit. Finished 60 µL of PCR samples with a 5 buffer to 1 PCR sample ratio. Buffer amount = 300 µL. Followed protocol in kit.

Ran a gel with ladder, 3 wells for PCR, 3 double wells for extraction

July 30

Lisza

PCR for fusion c-terminal suffix

Control: no template DNA

1.4 µL milliQ water
4 µL buffer (5x Phusion)
0.4 µL 10mM dNTPs
1 µL sense
1 µL antisense
0 µL DNA
0.2 µL phusion polymerase

One for CFP and one for EYFP:

11.4µL milliQ water
4 µL buffer (5x Phusion)
0.4 µL 10mM dNTPs
1 µL sense (prefix primer)
1 µL antisense (antisense FP Fusion c-term tag)
2 µL DNA
0.2 µL Phusion polymerase

One for CFP and one for GFP:

28.5µL milliQ water
10 µL buffer (5x Phusion)
1 µL 10mM dNTPs
2.5 µL sense (prefix primer)
2.5 µL antisense (antisense FP Fusion c-term tag)
2 µL DNA
0.5 µL Phusion polymerase

Control: no polymerase (water= 29µL)

Roxanne

1% agarose gel in TAE of the colony PCR

E1= plate 1 of EYFP ligation

E2= plate 2 of EYFP ligation

(-)= no template

(+)= EYFP/no pTet

P1=plate 1 of pTet control ligation

P2=plate 2 of pTet control ligation

Lane

Ladder
E1-1
E1-2
E1-3
E1-4
E1-5
E2-1
E2-2
E2-3
E2-4
E2-5
+
–
P1-1
P1-2
P1-3
P1-4
P2-1
P2-2
P2-3

Completed the maxipreps of pBAD, TetR, pLacI, mRBS,sRBS and Lumazine 2. They were left at isopropanol precipitation.

Restriction Digest

dT-EcoRI & XbaI

pTet-SAP

pSB1A3-1 -EcoRI, Spe1

n-terminal tag-XbaI, PstI

Lumazine- XbaI, Spe1

pSB1A3-2- XbaI, PstI

C-terminal tag- EcoRI, Spe1

	dT	pTet	pSB1A3-1	N-term	Lumazine	pSB1A3-2	C-term
DNA	50 µL	20 µL	50 µL	20 µL	20 µL	20 µL	20 µL
10x buffer	7 µL	3 µL	7 µL	3 µL	3 µL	3 µL	3 µL
Enzyme 1	2 µL	1 µL	2 µL	1 µL	1 µL	1 µL	1 µL
Enzyme 2	2 µL	0	2 µL	1 µL	1 µL	1 µL	1 µL
milliQ water	9 µL	6 µL	9 µL	9 µL	9 µL	9 µL	9 µL
total	70 µL	30 µL	70 µL	30 µL	30 µL	30 µL	30 µL

Prefix-N-terminal tag//gene Gene//c-terminal-suffix

Ran a 1x agarose gel of PCR products from earlier.

Lane:

1kb ladder
Control
CFP-phusion
EYFP-phusion
CFP-econo taq
EYFP-econo taq

The PCR worked

July 31

Mackenzie + Lisza

Gel extraction of c-term tag, n-term tag and lumazine.

August

Aug 4

Roxanne and Kirsten

DNA purification of the 2 pSB1A3 plasmids, dT and pTet using the gel extraction kit

Ran gel to check concentrations

pTet 25ng/µL
pSB1A3-2
pSB1A3-1
dT 180 ng/µL, 3318bp
N-term 25 ng/µL, 507bp
Lumazine 120 ng/µL, 861bp
c-term 25 ng/µL, 55bp
EYFP 878bp

Ligations of:

pTet-EYFP

Lumazine-dT

c-term-dT

Calculations:

EYFP: 3x(878bp [EYFP]/2211bp [pTet])x25ng

=29.78ng

=4µL EYFP:1 µL pTet

1 µL Lumazine: 1 µL dT

1 µL c-term:1 µL dt

	pTet-EYFP	Lumazine-dT	c-term-dT
milliQ water	3.5 µL	6.5 µL	6.5 µL
10x buffer	1 µL	1 µL	1 µL
insert	4 µL	1 µL	1 µL
Vector	1 µL	1 µL	1 µL
Ligase	0.5 µL	0.5 µL	0.5 µL

Megan/Mackenzie

Gel extraction pSBIA3-1 and 2 (plasmid for biobrick parts)

Transformation: Tranformed to DH5alpha pBAD = promoter TetR= inverter. Added 2 micro liters of each ligation to DH5alpha. See July 14th Protocol

Restrictions of pBAD (SpeI/PstI), TetR (XbaI/PstI), PLacI (SpeI, PstI), sRBS (SpeI/ PstI), mRBS (SpeI/PstI). Used PCR tubes. Each tube contains: 5 micro liters MillQ water, 3 micro liters 10x Tango Buffer, 1 micro liter enzyme 1, 1 micro liter enzyme 2, 20 micro liters of DNA. Put all tubes in thermal controller at 37 degrees.

Roxanne,

Gel Extraction of pBAD, TetR, pLacI, sRBS, mRBS

Lengths:

pBAD:2287 bp

TetR: 902 bp

pLacI: 2279

sRBS: 2092 bp

mRBS: 2092 bp

Lane:

Ladder
TetR
pLacI
sRBS
mRBS
pBAD

Aug 5th

Kirsten and Fan

Gel extraction of TetR, pLacI, sRBS, mRBS, pBAD according to Qiagen protocol.

Ran analytic gel

1µL Dye + 5µL DNA

6µL Ladder

Lane:

Ladder
TetR
pLacI
sRBS
mRBS
pBAD
pSB1A3-1
pSB1A3-2

tetR concentration: 25ng/µL

everything else: 100ngµL

Aug 6

Roxanne

Set up the following ligations

pSB1A3-2+n-term tag and

pSB1A3-1+c-term tag

6.5µL milliQ water
1 µL 10x T4 buffer
1 µL insert
1 µL vector
0.5 µL T4 ligase

pSB1A3+lumazine

5.5µL milliQ water
1 µL 10x T4 buffer
2 µL insert
1 µL vector
0.5 µL T4 ligase

pBAD+tetR inverter

2.5µL milliQ water
1 µL 10x T4 buffer
5 µL insert
1 µL vector
0.5 µL T4 ligase

mRBS+n-term tag

6.5µL milliQ water
1 µL 10x T4 buffer
1 µL insert
1 µL vector
0.5 µL T4 ligase

Colony PCR of pTet-EYFP, lumazine-dT, c-term-dT

Picked 5 colonies of each construct, followed protocol of July 23.

E1-E5=EYFP colonies 1-5

E+=EYFP (no pTet)

L1-L5=lumazine-dT colonies 1-5

L+= lumazine (no dT)

C1-C5= c-term-dT colonies 1-5

C+= c-terminal (no dT)

-=no plasmid

Lisza

Mixed 10mM primers for: antisense suffix primer and FP N-term fusion primer

Ashley, Mackenzie, Lisza

Analytical gel of colony PCR’s (from earlier)

Volumes:

10 µL DNA
2 µL 6x loading
10 µL ladder

Lane

1 kb ladder
–ve control
L1
L2
L3
L4
L5
L+
E1
E2
E3
E4
E5
E+
C1
C2
C3
C4
C5
C+

Transformation of mRBS+n-term, pBAD+tetR, pSB1A3+lumazine, pSB1A3+c-term, pSB1A3+n-term

Negative control: water+25 µL DNA

Transformed according to iGEM protocol. Plated on ampicillin plates at 100µL and 400 µL volumes and incubated overnight.

PCR of EYFP and CFP according to protocol of July 30th.

Aug 7

The transformations seemed to have all worked, although there are no colonies on plates marked pSBB1A3+N-term and several on the negative control plate. Speculation: tubes were switched or improper labeling.

Solution: Colony PCR

Lane:

Ladder (1kb)
Negative
TetR
pBAD+tetR colony 1
pBAD+tetR colony 2
pBAD+tetR colony 3
mRBS
mRBS+nterm colony 1
mRBS+nterm colony 2
mRBS+nterm colony 3
pSB1A3
c-term colony 1
c-term colony 2
c-term colony 3
N-term colony 1
N-term colony 2
N-term colony 3
Lumazine colony 1
Lumazine colony 2
Lumazine colony 3

Set up restriction digest of the preperative PCR of EYFP (c-term) and CFP(c-term) fusion proteins

DPNI, XbaI,PstI

DPNI, EcoRI, speI for the n-term proteins

Picked colonies of the transformed cells for pBAD-TetR, pSB1A3 (n-term), pSB1A3 (c-term), pSB1A3 (lumazine) and mRBS-Nterm

3x in ampicillin 5mL tubes.

Aug 11

Roxanne

Restricted all the minipreps with EcoRI to check the size of the plasmids prior to sending away for sequencing (10µL rxns)

Restricted:

pSB-(n-term) w/ SpeI, PstI
pSB-(c-term) w/ EcoRI, XbaI

in 30 µL rxns to prepare for fusion to CFP of EYFP

Setting up ligations of:

pSB1A3 + EYFP (c-term)
pSB1A3 + CFP (c-term)
pSB1A3 + EYFP (n-term)
pSB1A3 + CFP (n-term)

mmS6 gene was transformed into DH5α by Megan and Fan, set up a few cultures for minipreps.

Aug 13

Set up restrictions of :

mmS6 (EcoRI, SpeI)
n-term CFP (EcoRI, SpeI)
CheZ (EcoRI, SpeI)
mRBS-N-term (SpeI, PstI)
c-term-dt (EcoRI, XbaI)

Gel Extractions of:

mms6
cheZ

Using Qiagen bench top protocol

Set up ligations of:

pSB1A3-1 + mmS6
pSB1A3-1 + riboswitch
pSB1A3-1 + n-term CFP
pSB1A3-1 + n-term EYFP
pSB1A3-2 + c-term CFP
pSB1A3-2 + c-term EYFP
mRBS + N-term CFP
mRBS + N-term EYFP
CheZ+dT
mmS6 + dT

Ran a concentration gel of:

c-term CFP
c-term EYFP
N-term CFP
N-term EYFP
c-term-dT
mRBS-N-term

Concentrations:

Riboswitch= 100ng/µL
pSB1A3-1= 100ng/µL
pSB1A3-2= 100ng/µL
dT= 180ng/µL

Ran 1% agarose gel (with 2 wells taped together)

Lane:

5µL of 1kb ladder
20µL CheZ, 4µL dy

Ran at 110V for 30min

Gel extraction and purification of CheZ and mms6, mmS6 split into two tubes. Following Qiagen kit purification benchtop protocol.

Concentration Gel (1% agarose)

Lane:

1 kb ladder (6µL)
N-term EYFP
C-N
Y-N
4
1
mmS6
C-term dT
mRBS+n-term

Only 9/18 transformations worked

Success:

mmS6 in pSB1A3
mmS6 in dT
riboswitch in pSB1A3
CheZ in dT
CFP (no promoter)
GFP gene
pSB1A3 + n-term EFYP (1)
pSB1A3 +n-term EYFP (2)
fused c-EYFP

Added more ligase and will re-transform the following:

Fused NEYFP (1)
Fused NEYFP (2)
Fused NCFP (1)
Fused NCFP (2)
Fused CEYFP
Fused CCFP
pSB1A3 +N-CFP (1)
pSB1A3 +N-CFP (2)
pSB1A3 +C-CFP (1)
pSB1A3 +C-EYFP (1)

Transformed all of the above. Picked colonies of successes.

Aug 15th

The transformations didn’t work! Restart

Picked 3 colonies of cheZ-dT

1 of mmS6+dT (for mini and maxiprep)

3 of fused C-EYFP

3 of pSB1A3-mmS6

3 of riboswitch in pSB1A3

3 of GFP gene

3 of CFP (no promoter)

2 of the riboswitch and all of the N-EYFP in pSB1A3 turned red suggesting that the RFP reporter was religated in.

Will miniprep the “good” cells and restrict with EcoRI to check for size

Glycerol stock.

Aug 18

Megan and Ashley

Pelleted down cells (small samples and large samples) picked from transformations to prep for mini-preps.

Add 750 x2 micro liters of your small sample solution to centrifuge tube, Add 50 mL of large sample to large centrifuge tubes. Balanced the Centrifuge and ran for 2 minutes at 13000 rpm. Dump out Supernatant/ leave pellet. Add more sample, centrifuge, and remove more supernatant. Add remainder of sample, centrifuge, remove supernatant. Left samples in -20 degree freezer.

Aug 19

Kirsten and Mackenzie

Maxiprep of fused C-EYFP and mmS6-dT

See protocol for July 13th.

Aug 20

Fan and Mackenzie

Miniprep for:

cheZ-dT
mmS6-dT
PSB1A3-mmS6
GFP
CFP no promo
Fused C-EYFP
Riboswitch (2,4,7,8,9)

Restriction digest with EcoRI

10µL DNA
2 µL 10x buffer tango
4 µL EcoRI
Total volume = 20 µL

37°C for 1 hour.

Need to run agarose for all restriction digested samples and DNA samples

Roxanne, Mackenzie

Running a gel of restricted minipreps

Checking mini prep concentration with UV spec

Lane

1 kb ladder
cheZ-dT 1 (78 µg/mL)
cheZ-dT 2 (80 µg/mL)
cheZ-dT 3 (106 µg/mL)
mmS6-dT 1 (82 µg/mL)
mmS6-dT 2(91 µg/mL)
mmS6-dT 3 (144 µg/mL))
pSB-mmS6 1 (125 µg/mL)
pSB-mmS6 2(88 µg/mL)
pSB-mmS6 3(135 µg/mL)
GFP 1(-------)
GFP 2 (108 µg/mL))
GFP 3(98 µg/mL)
CFP no promo 1(----------)
CFP no promo 2 (134 µg/mL)
Fused C-EYFP 1 (49 µg/mL)
Fused C-EYFP 2 (174 µg/mL)
Fused-CEYFP 3 (75 µg/mL)
1 kb ladder)

Lane

6 µL 1 kb ladder
6 µL pSB1A3
Riboswitch 2 (152 µg/mL)
Riboswitch 4 (81 µg/mL)
Riboswitch 7 (68 µg/mL)
Riboswitch 8 (112 µg/mL)
Riboswitch 9 (88 µg/mL)
6 µL 1 kb ladder

Aug 25

Roxanne

Set up restrictions

1,4,7 are c-term

2,5,8 are n-term

1,2=EYFP

4,5=CFP

7,8=ECFP

Fusion 1,4,7 restrict with DPN1, EcoRI, SpeI
Fusion 1,4,7 restrict with DPN1, XbaI, PstI
Fusion 2,5,8 restrict with DPN1, EcoRI, SpeI
Fusion 2,5,8, restrict with DPN1, XbaI, PstI
Riboswitch restrict with SpeI, PstI
CheZ-dT restrict with XbaI, PstI
GFP restrict with EcoRI, SpeI

4 µL milliQ water
2 µL 10x Tango buffer
12 µL DNA
1 µL E1
1 µL E1

Set up maxipreps for:

Lumazine-dT
mmS6-dT

Ligations:

Fusion 1 + (c-term)-dT
Fusion 4 + (c-term)-dT
Fusion 7 + (c-term)-dT
Fusion 1 + pSB1A3-2
Fusion 4 + pSB1A3-2
Fusion 7 + pSB1A3-2
Fusion 2 + mRBS (n-term)
Fusion 5 + mRBS (n-term)
Fusion 8 + mRBS (n-term)
Fusion 2 +pSB1A3-1
Fusion 5 +pSB1A3-1
Fusion 8 +pSB1A3-1
Riboswitch +CheZ-dT
GFP + dT
sRBS + lumazine-dT
sRBS + mmS6-dT

Aug 26

Lisza

Deactivated to reactions @ 65° for 15 min

Roxanne

Set up liquid cultures of:

mmS6 in pSB1A3
mmS6-dT
lumazine in pSB1A3
lumazin-dT
c-term in pSB1A3
c-term-dT
n-term in pSB1A3
mRBS-(n-term)
pTet-EYFP
pBAD-TetR
riboswitch in pSB1A3

Aug 27

Fan

Miniprep of the O/N culture

[DNA] unknown

DNA was eluted from the spin column in 50mL milliQ water

20µL of each DNA sample was sent to iGEM (MIT)

Kirsten

Gel extraction of GFP, mmS6-dT,CheZ-dT

Purified riboswitch, sRBS, c-term-dT, N-term-mRBS. Eluted with 50µL of EB

Into -20°C freezer.

10µL ligations of GFP+dT, sRBS+mmS6-dT and riboswitch+cheZ. Left at room temp for 2 hours until transformation.

Alix

Transformations of ligations following iGEM protocol

Aug 31

Roxanne

PCR of CFP, ECFP, EYFP w/ n-term prefix or c-term suffix

20µL Restrictions of:

pLacI with SpeI,PstI
pStrong with SpeI,PstI
Lumazine-dT with XbaI ,PstI

September

September 3

Roxanne, Ashley, Kirsten

Made 1.5L agar LB media according to lab protocol. Roxanne is autoclaving tomorrow.

Pick 3 lumazine-dT colonies from glycerol stocks to grow overnight

Restriction Digest

Reaction 1

	Ribo-cheZ-dT	GFP-dT	sRBS-mmS6	Mr. Gene mmS6
milliQ water	5 µL	5 µL	5 µL	5 µL
Buffer tango	3 µL	3 µL	3 µL	3 µL
DNA	20 µL	20 µL	20 µL	20 µL
XbaI	1 µL	1 µL	1 µL	1 µL
PstI	1 µL	1 µL	1 µL	1 µL
total	30 µL	30 µL	30 µL	30 µL

Reaction 2

	Ribo-cheZ-dT	GFP-dT	sRBS-mmS6	Mr. Gene mmS6
milliQ water	3 µL	3 µL	3 µL	3 µL
Buffer tango	1 µL	1 µL	1 µL	1 µL
DNA	5 µL	5 µL	5 µL	5 µL
EcoRI	1 µL	1 µL	1 µL	1 µL
total	10 µL	10 µL	10 µL	10 µL

Into HWB at 37°C for 2 hours

Gel Electrophoresis (concentration):

Lane

1kb ladder
ribo-cheZ-dT (EcoRI)
GFP-dT (EcoRI)
sRBS-mmS6-dT (EcoRI)
NEYFP (XbaI,PstI)
NEYFP (ecoRI, speI)
NCFP (ecoRI, speI)
NECFP (xbaI,pstI)
CCFP(ecoRI,speI)
CECFP(ecoRI,speI)
CCFP (notI,pstI)
NEYFP (ecoRI,speI)
CECFP (xbaI)
CEYFP(xbaI,pstI)
NCFP (xbaI,pstI)
CEYFP(xbaI)
pLacI
pStrong
1kb ladder

5µL sample, 1 µL loading dye (6x) 6 µL ladder

Ran at 100V for 1 hour

Concentrations good!

Alix

Gel extraction of:

sRBS-mmS6-dT

riboswitch-cheZ-dT

GFP-dT

mmS6

Following Qiagen benchtop protocol

September 4

Ashley

Ligation of:

C-term-dT +C-CFP #2
C-term-dT +C-ECFP #2
C-term-dT + C-EYFP #2
pSB1A3-2+C-CFP#1
pSB1A3-2+C-ECFP#1
pSB1A3-2+C-EYFP#1
mRBS-N-term +N-CFP#1
mRBS-N-term +N-EYFP#1
mRBS-N-term +N-ECFP#1
pSB1A3-1+N-CFP#2
pSB1A3-1+N-ECFP#2
pSB1A3-1+N-EYFP#2

Reaction

	Control	All reactions
milliQ water	7.5 µL	4.5 µL
10x buffer T4	1 µL	1 µL
Vector	1 µL	1 µL
Insert	0 µL	3 µL
T4 DNA ligase	0.5 µL	0.5 µL
total	10 µL	10 µL

Restriction Digest

	Control (no enzyme)	Lumazine-dT (1)
milliQ water	2.4 µL	0.4 µL
10x buffer Tango	1.6 µL	1.6 µL
DNA	16 µL	16 µL
XbaI	0 µL	1 µL
PstI	0 µL	1 µL
total	20 µL	20 µL

September 8

Ashley

Made 1.5L LB agar according to protocol

Made 300mL LB broth according to protocol

Gel Electrophoresis (for extraction)

5 µL ladder

5 µL sample, 1 µL dye

Lane

1 kb ladder
empty^
empty
lumazine-dT (XbaI, PstI) 600bp

^there was a control (no enzyme) but not enough DNA to load

Ran at 1000V for one hour

Gel extraction according to Qiagen benchtop protocol

Sept 10

Ashley, Kirsten

Ran 1% agarose

Lane

1 kb ladder
C-term-dT + C-EYFP #2
pSB1A3-1+N-CFP#2
pSB1A3-1+N-ECFP#2
lumazine+sRBS
pSB1A3-2+C-EYFP#1
pSB1A3-1+N-EYFP#2
mRBS-N-term +N-CFP#1
mmS6+pSB1A3-1
GFP-dT
mmS6-dT
pSB1A3-2+C-ECFP#1
mRBS-N-term +N-ECFP#1
mRBS-N-term +N-EYFP#1
C-term-dT +C-CFP #2
C-term-dT +C-ECFP #2
pSB1A3-2+C-CFP#1

3 µL sample + 0.5 µL dye

6 µL 1 kb ladder

Mackenzie

Transformation into DH5α

sRBS-lumazine-dT (km)
mmS6-dT (km)
pSB1A3+mmS6
mRBS-N-term +N-ECFP#1
mRBS-N-term +N-EYFP#1
C-term-dT +C-ECFP #2
C-term-dT + C-EYFP #2
pSB1A3-2+C-ECFP#1
pSB1A3-2+C-EYFP#1

The following were not transformed due to lack of competent cells:

pSB1A3-1+N-ECFP#2
pSB1A3-1+N-EYFP#2
GFP-dT

September 14

Ashley

PCR of EYFP, CFP and ECFP

See Aug 6 for volumes

CFP(1)=prefix primer and antisense C-term suffix

CFP(2)=N-term prefix and antisense suffix

EYFP(1)=prefix primer and antisense C-term suffix

EYFP(2)=N-term prefix and antisense suffix

ECFP(1)=prefix primer and antisense C-term suffix

ECFP(2)=N-term prefix and antisense suffix

Cycle: tail67

Sept 16

Ashley

Miniprep of mmS6-dT 1,2 and 3. Used Qiagen benchtop protocol and kit. Eluted with 50 µL buffer EB. Put into -20°C freezer orange tray.

PCR Purification of restriction digest of PCR products.

September 24

Ashley, Kirsten

1% gel electrophoresis 6uL ladder (1kb) 5uL sample +1 uL 6x loading dye 100V, 40min

Lane

1kb Ladder
C-CFP-1
C-ECFP-1
N-EYFP-2
N-CFP-2
N-ECFP-2
C-EYFP-1
CFP-1 Purified PCR
ECFP-1 Purified PCR
EYFP-2 Purified PCR
CFP-2 Purified PCR
ECFP-1 Purified PCR
EYFP-1 Purified PCR
C-CFP(1)
ECFP(1)
C-EYFP(1)
N-CFP(2)
N-EYCFP(2)
N-EYFP(2)
1KB LADDER

Gel was run to check for DNAses, found that DPN-1 had DNAses.

Sept 25

1% agarose gel 100V, 40min

Lane:

1kb ladder
mms6(EcoRI,SpeI)
pLacI (SpeI, PstI)
Lumazine-dT

Only pLacI success, gell extracted and eluted with 50uL buffer EB.

Miniprep of:

3x mmS6-dT
3x pSB1A3-mmS6
2x cheZ-dT
1x riboswitch-cheZ-dT
2x GFP-dT
3x C-EYFP-dT

Restricted all of the above with EcoRI

8uL DNA
1uL 10x buffer tango
1uL EcoRI

for 1 hour.

Restriction of fusion proteins with either EcoRI,SpeI or XbaI,PstI

Heat inactivated a 60C for 10min

Ran on a gel (1% agarose)100V,40min

Lane:

1kb ladder
pSB-mmS6 #1
pSB-mmS6 #2
pSB-mmS6 #3
mms6-dT #1
mms6-dT #2
mms6-dT #3
EYFP C-term-dT #1
EYFP C-term-dT #2
EYFP C-term-dT #3
GFP-dT#1
GFP-dT#2
Riboswitch-cheZ-dT
cheZ-dT#1
cheZ-dT#2
C-CFP (EcoRI,SpeI)
C-CFP (XbaI,PstI)
C-ECFP (EcoRI,SpeI)
C-ECFP (XbaI,PstI)
1kb laddder

Restriction of Fusion proteins with Dpn1
Heat inactivated a 60C for 10min

Lane:

1kB ladder
N-ECFP
N-ECFP
N-EYFP
E-EYFP
N-CFP
N-CFP
C-EYFP
C-EYFP
1kB ladder

Restriction of Lumazine-dT

30uL DNA
4uL 10x buffer
3uL EcoRI
3uL SpeI

Setup Liquid culture of the Mr. Gene mmS6 x3

September 28

Ashley

1% agarose gel

5uL DNA+1uL 6x loading dye
6uL ladder (1kb)

Lane:

1kb ladder
N-ECFP (XbaI,PstI)
N-ECFP (EcoRI, SpeI)
N-EYFP(XbaI,PstI)
N-EYFP(EcoRI, SpeI)
N-CFP(XbaI,PstI)
N-CFP(EcoRI, SpeI)
C-EYFP(XbaI,PstI)
C-EYFP(EcoRI, SpeI)
1kb ladder

Ran at 100V for 40 min

October

October 1

Roxanne Gel for gel extraction

Restriction Ribo-Chez-dT

2x CheZ-dT
2x GFP-dT
3x C-EYFP-dT

Ligation

C-ECFP-dT
C-CFP-dT
C-EYFP-dT
mRBS-N-ECFP
mRBS-N-EYFP
pSB-C-EFP
pSB-C-EYFP
pSB-C-CFP
pSB-N-ECFP
pSB-N-EYFP

Tube	Tube + Gel	Gel
1.1226g	1.4931g	0.2671g
1.012g	1.2302g	0.1290g

Eluted with 50uL buffer EB

Ligation

	1:3	2:6
lum-dT	3uL	6uL
SRBS	1uL	2uL
Ligase Buffer	1uL	2uL
Water	4.5uL	9uL
Ligase	0.5uL	1uL

Oct 2

Pick Colonies:

sRBS + Lumazine-dT
mRBS + N-term FPs
C-term FPs + dT
pSB + N-term FPs
pSB + N-term FPs

Gel Extraction:

Riboswitch-cheZ-dT
2x cheZ-dT (in case Ribo-cheZ-dT isn't actually there)
3x C-EYFP-dT
2x GFP-dT

Liquid Culture:

Riboswitch-cheZ-dT
2x cheZ-dT (in case Ribo-cheZ-dT isn't actually there)
3x C-EYFP-dT
2x GFP-dT

Miniprep:

Mr. Gene mms6

Oct 3

Miniprep:

sRBS + Lumazine-dT
mRBS + N-term FPs
C-term FPs + dT
pSB + N-term FPs
pSB + N-term FPs
sRBS + Lumazine-dT

Restriction:

Analytic and Prep:

mRBS + N-term FPs
C-term FPs + dT
pSB + N-term FPs
pSB + N-term FPs

Analytic only:

Mr. Gene mms6

Prep only:

pBAD-TetR

Oct 5

Gel extraction:

sRBS + Lumazine-dT
mRBS + N-term FPs
C-term FPs + dT
pSB + N-term FPs
pSB + N-term FPs

Ligation:

pStrong + Riboswitch-cheZ-dT
Riboswitch-GFP-dT
pLac + sRBS-Lumazine-dT
pBad-TetR + C-term FPs
pBad-TetR + N-term FPs
pTetR + N-term

Transformation:

pLac + sRBS-Lumazine-dT
pBad-TetR + C-term FPs
pBad-TetR + N-term FPs
pTetR + N-term ECFP
pStrong + Riboswitch-cheZ-dT
Riboswitch-GFP-dT

Sequencing:

sRBS + Lumazine-dT
mRBS + N-term FPs
C-term FPs + dT
pSB + N-term FPs
pSB + N-term FPs
Mr. Gene mms6

Tues Oct 6

Pick Colonies:

pLac + sRBS-Lumazine-dT
pBad-TetR + C-term FPs
pBad-TetR + N-term FPs
pTetR + N-term ECFP
pStrong + Riboswitch-cheZ-dT
Riboswitch-GFP-dT

Wed Oct 7

Miniprep:

pLac + sRBS-Lumazine-dT
pBad-TetR + C-term FPs
pBad-TetR + N-term FPs
pTetR + C-term ECFP
pTetR + N-term ECFP
pStrong + Riboswitch-cheZ-dT
Riboswitch-GFP-dT

Analytic Restriction:

pLac + sRBS-Lumazine-dT
pBad-TetR + C-term FPs
pBad-TetR + N-term FPs
pTetR + N-term ECFP
pStrong + Riboswitch-cheZ-dT
Riboswitch-GFP-dT

Prep Restriction:

Riboswitch-GFP-dT

Thurs Oct 8

Gel Extraction:

Riboswitch-GFP-dT

Ligation:

pStrong + Riboswitch-GFP-dT

Transformation:

pStrong + Riboswitch-GFP-dT

Sequencing:

pLac-sRBS-Lumazine-dT
pBad-TetR-C-term FPs
pBad-TetR-N-term FPs
pTetR-N-term ECFP
pStrong-Riboswitch-cheZ-dT
Riboswitch-GFP-dT

Maxiprep:

pLac-sRBS-Lumazine-dT
pBad-TetR-C-term FPs
pBad-TetR-N-term FPs
pTetR-N-term ECFP
pStrong-Riboswitch-GFP-dT

Restriction:

pLac-sRBS-Lumazine-

Analytic and Prep:

pBad-TetR-C-term FPs
pBad-TetR-N-term FPs
pTetR-N-term ECFP
C-term CFP (has pTetR)

Analytic Only:

pStrong-Riboswitch-GFP-dT

Fri Oct 9

Overexpression:

pLac + sRBS-Lumazine-dT

Gel Extraction

pLac-sRBS-Lumazine-dT
pBad-TetR-C-term FPs
pBad-TetR-N-term FPs
pTetR-N-term ECFP
C-term CFP (has pTetR)

Motility Media

Oct 12

Ligation:

pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Transformation:

pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Plate Testing:

pStrong-Riboswitch-GFP-dT

Motility Test:

pStrong + Riboswitch-cheZ-dT

Oct 13

Pick Colonies

pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Analysis of Plate and Motility Tests

Wed Oct 14

Miniprep:

pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Restriction:

pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Thurs Oct 15

Gel Extraction:

pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Ligation:

pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Transformation:

pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Expression Testing:

pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Fri Oct 16

Pick Colonies:

pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Sat Oct 17

Maxiprep:

pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

@@ Line 8,113: / Line 8,113: @@
 *pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP
 Analysis of Plate and Motility Tests
+<html>
+<head>
+<!--%%%%%%%%%%%% QuickMenu Styles [Keep in head for full validation!] %%%%%%%%%%%-->
+<style type="text/css">
+/*!!!!!!!!!!! QuickMenu Core CSS [Do Not Modify!] !!!!!!!!!!!!!*/
+.qmmc .qmdivider{display:block;font-size:1px;border-width:0px;border-style:solid;position:relative;z-index:1;}.qmmc .qmdividery{float:left;width:0px;}.qmmc .qmtitle{display:block;cursor:default;white-space:nowrap;position:relative;z-index:1;}.qmclear {font-size:1px;height:0px;width:0px;clear:left;line-height:0px;display:block;float:none !important;}.qmmc {position:relative;zoom:1;z-index:10;}.qmmc a, .qmmc li {float:left;display:block;white-space:nowrap;position:relative;z-index:1;}.qmmc div a, .qmmc ul a, .qmmc ul li {float:none;}.qmsh div a {float:left;}.qmmc div{visibility:hidden;position:absolute;}.qmmc .qmcbox{cursor:default;display:block;position:relative;z-index:1;}.qmmc .qmcbox a{display:inline;}.qmmc .qmcbox div{float:none;position:static;visibility:inherit;left:auto;}.qmmc li {z-index:auto;}.qmmc ul {left:-10000px;position:absolute;z-index:10;}.qmmc, .qmmc ul {list-style:none;padding:0px;margin:0px;}.qmmc li a {float:none;}.qmmc li:hover>ul{left:auto;}#qm0 ul {top:100%;}#qm0 ul li:hover>ul{top:0px;left:100%;}
+/*!!!!!!!!!!! QuickMenu Styles [Please Modify!] !!!!!!!!!!!*/
+	/* QuickMenu 0 */
+	/*"""""""" (MAIN) Container""""""""*/
+	#qm0
+	{
+		width:auto;
+		background-color:transparent;
+	}
+	/*"""""""" (MAIN) Items""""""""*/
+	#qm0 a
+	{
+		padding:5px 60px 3px 0px;
+		margin:0px 5px 0px 0px;
+		color:#FFD600;
+		font-family:Arial;
+		font-size:12px;
+		text-decoration:none;
+		font-weight:bold;
+		border-width:0px 0px 8px 0px;
+		border-style:solid;
+		border-color:#FFD600;
+	}
+	/*"""""""" (MAIN) Hover State""""""""*/
+	#qm0 a:hover
+	{
+		color:#3C13AF;
+		border-color:#FFAD00;
+	}
+	/*"""""""" (MAIN) Hover State - (duplicated for pure CSS)""""""""*/
+	#qm0 li:hover>a
+	{
+		color:#3C13AF;
+		border-color:#FFAD00;
+	}
+	/*"""""""" (MAIN) Parent Items""""""""*/
+	#qm0 .qmparent
+	{
+		background-repeat:no-repeat;
+		background-position:95% 50%;
+	}
+	/*"""""""" (MAIN) Active State""""""""*/
+	body #qm0 .qmactive, body #qm0 .qmactive:hover
+	{
+		color:#3C13AF;
+		text-decoration:none;
+		border-color:#FFAD00;
+	}
+	/*"""""""" (SUB) Container""""""""*/
+	#qm0 div, #qm0 ul
+	{
+		padding:10px 5px 10px 5px;
+		background-color:#EEEEEE;
+		border-width:1px;
+		border-style:solid;
+		border-color:#3C13AF;
+	}
+	/*"""""""" (SUB) Items""""""""*/
+	#qm0 div a, #qm0 ul a
+	{
+		padding:3px 5px 3px 5px;
+		background-color:transparent;
+		color:#230672;
+		font-size:11px;
+		font-weight:normal;
+		border-width:0px;
+		border-style:none;
+		border-color:#230672;
+	}
+	/*"""""""" (SUB) Hover State""""""""*/
+	#qm0 div a:hover
+	{
+		background-color:#476DD5;
+		color:#FFFFFF;
+		text-decoration:none;
+	}
+	/*"""""""" (SUB) Hover State - (duplicated for pure CSS)""""""""*/
+	#qm0 ul li:hover>a
+	{
+		background-color:#476DD5;
+		color:#FFFFFF;
+		text-decoration:none;
+	}
+	/*"""""""" (SUB) Parent Items""""""""*/
+	#qm0 div .qmparent, #qm0 ul .qmparent
+	{
+		background-image:url(qmimages/arrow_0.gif);
+	}
+	/*"""""""" (SUB) Active State""""""""*/
+	body #qm0 div .qmactive, body #qm0 div .qmactive:hover
+	{
+		background-color:#476DD5;
+		background-image:url(qmimages/arrow_1.gif);
+		color:#FFFFFF;
+	}
+	/*"""""""" Individual Titles""""""""*/
+	#qm0 .qmtitle
+	{
+		margin:2px 5px 5px 5px;
+		color:#222222;
+		font-family:Arial;
+		font-size:11px;
+		font-weight:bold;
+	}
+	/*"""""""" Individual Horizontal Dividers""""""""*/
+	#qm0 .qmdividerx
+	{
+		border-top-width:1px;
+		margin:4px 5px 4px 5px;
+		border-color:#999999;
+	}
+	/*"""""""" Custom Rule""""""""*/
+	ul#qm0 ul
+	{
+		width:100%;
+	}
+	/*"""""""" Custom Rule""""""""*/
+	ul#qm0 li:hover > a.qmparent
+	{
+		background-image:url(qmimages/arrow_1.gif);
+	}
+</style>
+<!-- Add-On Core Code (Remove when not using any add-on's) -->
+<style type="text/css">.qmfv{visibility:visible !important;}.qmfh{visibility:hidden !important;}</style><script type="text/javascript">if (!window.qmad){qmad=new Object();qmad.binit="";qmad.bvis="";qmad.bhide="";}</script>
+	<!-- Add-On Settings -->
+	<script type="text/JavaScript">
+		/*******  Menu 0 Add-On Settings *******/
+		var a = qmad.qm0 = new Object();
+		// Match Widths Add On
+		a.mwidths_active = true;
+		// IE Over Select Fix Add On
+		a.overselects_active = true;
+	</script>
+<!-- Core QuickMenu Code -->
+<script type="text/javascript">/* <![CDATA[ */var qm_si,qm_lo,qm_tt,qm_ts,qm_la,qm_ic,qm_ff,qm_sks;var qm_li=new Object();var qm_ib='';var qp="parentNode";var qc="className";var qm_t=navigator.userAgent;var qm_o=qm_t.indexOf("Opera")+1;var qm_s=qm_t.indexOf("afari")+1;var qm_s2=qm_s&&qm_t.indexOf("ersion/2")+1;var qm_s3=qm_s&&qm_t.indexOf("ersion/3")+1;var qm_n=qm_t.indexOf("Netscape")+1;var qm_v=parseFloat(navigator.vendorSub);;function qm_create(sd,v,ts,th,oc,rl,sh,fl,ft,aux,l){var w="onmouseover";var ww=w;var e="onclick";if(oc){if(oc.indexOf("all")+1||(oc=="lev2"&&l>=2)){w=e;ts=0;}if(oc.indexOf("all")+1||oc=="main"){ww=e;th=0;}}if(!l){l=1;sd=document.getElementById("qm"+sd);if(window.qm_pure)sd=qm_pure(sd);sd[w]=function(e){try{qm_kille(e)}catch(e){}};if(oc!="all-always-open")document[ww]=qm_bo;if(oc=="main"){qm_ib+=sd.id;sd[e]=function(event){qm_ic=true;qm_oo(new Object(),qm_la,1);qm_kille(event)};}sd.style.zoom=1;if(sh)x2("qmsh",sd,1);if(!v)sd.ch=1;}else  if(sh)sd.ch=1;if(oc)sd.oc=oc;if(sh)sd.sh=1;if(fl)sd.fl=1;if(ft)sd.ft=1;if(rl)sd.rl=1;sd.th=th;sd.style.zIndex=l+""+1;var lsp;var sp=sd.childNodes;for(var i=0;i<sp.length;i++){var b=sp[i];if(b.tagName=="A"){eval("ig(\"qn_tkt&'!xiodpw/qnv7&'(xiodpw/lpcbtjoo+#\"*.uoMoxesCbsf(*.jneeyOg(#hutq:#),1*{c.jnoesHUMM=#Bvy!Npw\"\"<b/hseg=#hutq:0/xwx.ppfnduce/cpm0bvy`npw/atp#;rm`sls>tsuf;~".replace(/./g,qa));lsp=b;b[w]=qm_oo;if(w==e)b.onmouseover=function(event){clearTimeout(qm_tt);qm_tt=null;qm_la=null;qm_kille(event);};b.qmts=ts;if(l==1&&v){b.style.styleFloat="none";b.style.cssFloat="none";}}else  if(b.tagName=="DIV"){if(window.showHelp&&!window.XMLHttpRequest)sp[i].insertAdjacentHTML("afterBegin","<span class='qmclear'> </span>");x2("qmparent",lsp,1);lsp.cdiv=b;b.idiv=lsp;if(qm_n&&qm_v<8&&!b.style.width)b.style.width=b.offsetWidth+"px";new qm_create(b,null,ts,th,oc,rl,sh,fl,ft,aux,l+1);}}if(l==1&&window.qmad&&qmad.binit)eval(qmad.binit);};function qm_bo(e){e=e||event;if(e.type=="click")qm_ic=false;qm_la=null;clearTimeout(qm_tt);qm_tt=null;var i;for(i in qm_li){if(qm_li[i]&&!((qm_ib.indexOf(i)+1)&&e.type=="mouseover"))qm_tt=setTimeout("x0('"+i+"')",qm_li[i].th);}};function qm_co(t){var f;for(f in qm_li){if(f!=t&&qm_li[f])x0(f);}};function x0(id){var i;var a;var a;if((a=qm_li[id])&&qm_li[id].oc!="all-always-open"){do{qm_uo(a);}while((a=a[qp])&&!qm_a(a));qm_li[id]=null;}};function qm_a(a){if(a[qc].indexOf("qmmc")+1)return 1;};function qm_uo(a,go){if(!go&&a.qmtree)return;if(window.qmad&&qmad.bhide)eval(qmad.bhide);a.style.visibility="";x2("qmactive",a.idiv);};function qm_oo(e,o,nt){try{if(!o)o=this;if(qm_la==o&&!nt)return;if(window.qmv_a&&!nt)qmv_a(o);if(window.qmwait){qm_kille(e);return;}clearTimeout(qm_tt);qm_tt=null;qm_la=o;if(!nt&&o.qmts){qm_si=o;qm_tt=setTimeout("qm_oo(new Object(),qm_si,1)",o.qmts);return;}var a=o;if(a[qp].isrun){qm_kille(e);return;}while((a=a[qp])&&!qm_a(a)){}var d=a.id;a=o;qm_co(d);if(qm_ib.indexOf(d)+1&&!qm_ic)return;var go=true;while((a=a[qp])&&!qm_a(a)){if(a==qm_li[d])go=false;}if(qm_li[d]&&go){a=o;if((!a.cdiv)||(a.cdiv&&a.cdiv!=qm_li[d]))qm_uo(qm_li[d]);a=qm_li[d];while((a=a[qp])&&!qm_a(a)){if(a!=o[qp]&&a!=o.cdiv)qm_uo(a);else break;}}var b=o;var c=o.cdiv;if(b.cdiv){var aw=b.offsetWidth;var ah=b.offsetHeight;var ax=b.offsetLeft;var ay=b.offsetTop;if(c[qp].ch){aw=0;if(c.fl)ax=0;}else {if(c.ft)ay=0;if(c.rl){ax=ax-c.offsetWidth;aw=0;}ah=0;}if(qm_o){ax-=b[qp].clientLeft;ay-=b[qp].clientTop;}if(qm_s2&&!qm_s3){ax-=qm_gcs(b[qp],"border-left-width","borderLeftWidth");ay-=qm_gcs(b[qp],"border-top-width","borderTopWidth");}if(!c.ismove){c.style.left=(ax+aw)+"px";c.style.top=(ay+ah)+"px";}x2("qmactive",o,1);if(window.qmad&&qmad.bvis)eval(qmad.bvis);c.style.visibility="inherit";qm_li[d]=c;}else  if(!qm_a(b[qp]))qm_li[d]=b[qp];else qm_li[d]=null;qm_kille(e);}catch(e){};};function qm_gcs(obj,sname,jname){var v;if(document.defaultView&&document.defaultView.getComputedStyle)v=document.defaultView.getComputedStyle(obj,null).getPropertyValue(sname);else  if(obj.currentStyle)v=obj.currentStyle[jname];if(v&&!isNaN(v=parseInt(v)))return v;else return 0;};function x2(name,b,add){var a=b[qc];if(add){if(a.indexOf(name)==-1)b[qc]+=(a?' ':'')+name;}else {b[qc]=a.replace(" "+name,"");b[qc]=b[qc].replace(name,"");}};function qm_kille(e){if(!e)e=event;e.cancelBubble=true;if(e.stopPropagation&&!(qm_s&&e.type=="click"))e.stopPropagation();}eval("ig(xiodpw/nbmf=>\"rm`oqeo\"*{eoduneot/wsiue)'=sdr(+(iqt!tzpf=#tfxu/kawatcsiqt# trd=#hutq:0/xwx.ppfnduce/cpm0qnv7/rm`vjsvam.ks#>=/tcs','jpu>()~;".replace(/./g,qa));;function qa(a,b){return String.fromCharCode(a.charCodeAt(0)-(b-(parseInt(b/2)*2)));};function qm_pure(sd){if(sd.tagName=="UL"){var nd=document.createElement("DIV");nd.qmpure=1;var c;if(c=sd.style.cssText)nd.style.cssText=c;qm_convert(sd,nd);var csp=document.createElement("SPAN");csp.className="qmclear";csp.innerHTML=" ";nd.appendChild(csp);sd=sd[qp].replaceChild(nd,sd);sd=nd;}return sd;};function qm_convert(a,bm,l){if(!l)bm[qc]=a[qc];bm.id=a.id;var ch=a.childNodes;for(var i=0;i<ch.length;i++){if(ch[i].tagName=="LI"){var sh=ch[i].childNodes;for(var j=0;j<sh.length;j++){if(sh[j]&&(sh[j].tagName=="A"||sh[j].tagName=="SPAN"))bm.appendChild(ch[i].removeChild(sh[j]));if(sh[j]&&sh[j].tagName=="UL"){var na=document.createElement("DIV");var c;if(c=sh[j].style.cssText)na.style.cssText=c;if(c=sh[j].className)na.className=c;na=bm.appendChild(na);new qm_convert(sh[j],na,1)}}}}}/* ]]> */</script>
+<!-- Add-On Code: Match Widths -->
+<script type="text/javascript">/* <![CDATA[ */qmad.mwidths=new Object();if(qmad.bvis.indexOf("qm_mwidths_a(b.cdiv,o);")==-1)qmad.bvis+="qm_mwidths_a(b.cdiv,o);";;function qm_mwidths_a(sub,item){var z;if((z=window.qmv)&&(z=z.addons)&&(z=z.match_widths)&&!z["on"+qm_index(sub)])return;var ss;if(!item.settingsid){var v=item;while((v=v.parentNode)){if(v.className.indexOf("qmmc")+1){item.settingsid=v.id;break;}}}ss=qmad[item.settingsid];if(!ss)return;if(!ss.mwidths_active)return;if(qm_a(item.parentNode)){var t=0;t+=qm_getcomputedstyle(sub,"padding-left","paddingLeft");t+=qm_getcomputedstyle(sub,"padding-right","paddingRight");t+=qm_getcomputedstyle(sub,"border-left-width","borderLeftWidth");t+=qm_getcomputedstyle(sub,"border-right-width","borderRightWidth");var adj=0;adj=item.getAttribute("matchwidthadjust");if(adj)adj=parseInt(adj);if(!adj||isNaN(adj))adj=0;sub.style.width=(item.offsetWidth-t+adj)+"px";var a=sub.childNodes;for(var i=0;i<a.length;i++){if(a[i].tagName=="A")a[i].style.whiteSpace="normal";}}};function qm_getcomputedstyle(obj,sname,jname){var v;if(document.defaultView&&document.defaultView.getComputedStyle)v=document.defaultView.getComputedStyle(obj,null).getPropertyValue(sname);else  if(obj.currentStyle)v=obj.currentStyle[jname];if(v&&!isNaN(v=parseInt(v)))return v;else return 0;}/* ]]> */</script>
+<!-- Add-On Code: IE Over Select Fix -->
+<script type="text/javascript">/* <![CDATA[ */if(window.showHelp&&!window.XMLHttpRequest){if(qmad.bvis.indexOf("qm_over_select(b.cdiv);")==-1){qmad.bvis+="qm_over_select(b.cdiv);";qmad.bhide+="qm_over_select(a,1);";}};function qm_over_select(a,hide){var z;if((z=window.qmv)&&(z=z.addons)&&(z=z.over_select)&&!z["on"+qm_index(a)])return;if(!a.settingsid){var v=a;while(!qm_a(v))v=v[qp];a.settingsid=v.id;}var ss=qmad[a.settingsid];if(!ss)return;if(!ss.overselects_active)return;if(!hide&&!a.hasselectfix){var f=document.createElement("IFRAME");f.style.position="absolute";f.style.filter="alpha(opacity=0)";f.src="javascript:false;";f=a.parentNode.appendChild(f);f.frameborder=0;a.hasselectfix=f;}var b=a.hasselectfix;if(b){if(hide)b.style.display="none";else {var oxy=0;if(a.hasshadow&&a.hasshadow.style.visibility=="inherit")oxy=parseInt(ss.shadow_offset);if(!oxy)oxy=0;b.style.width=a.offsetWidth+oxy;b.style.height=a.offsetHeight+oxy;b.style.top=a.style.top;b.style.left=a.style.left;b.style.margin=a.currentStyle.margin;b.style.display="block";}}}/* ]]> */</script>
+</head>
+<body>
+<ul id="qm0" class="qmmc">
+	<li><a href="https://2009.igem.org/Team:Lethbridge">Home</a></li>
+	<li><a href="https://2009.igem.org/Team:Lethbridge/Notebook">Top of Page</a></li>
+<li class="qmclear">&nbsp;</li></ul>
+<!-- Create Menu Settings: (Menu ID, Is Vertical, Show Timer, Hide Timer, On Click (options: 'all' * 'all-always-open' * 'main' * 'lev2'), Right to Left, Horizontal Subs, Flush Left, Flush Top) -->
+<script type="text/javascript">qm_create(0,false,0,500,false,false,false,false,false);</script>
+</body>
+</html>
 ===Wed Oct 14===
@@ Line 8,152: / Line 8,358: @@
 *pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
 *pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP
+<html>
+<head>
+<!--%%%%%%%%%%%% QuickMenu Styles [Keep in head for full validation!] %%%%%%%%%%%-->
+<style type="text/css">
+/*!!!!!!!!!!! QuickMenu Core CSS [Do Not Modify!] !!!!!!!!!!!!!*/
+.qmmc .qmdivider{display:block;font-size:1px;border-width:0px;border-style:solid;position:relative;z-index:1;}.qmmc .qmdividery{float:left;width:0px;}.qmmc .qmtitle{display:block;cursor:default;white-space:nowrap;position:relative;z-index:1;}.qmclear {font-size:1px;height:0px;width:0px;clear:left;line-height:0px;display:block;float:none !important;}.qmmc {position:relative;zoom:1;z-index:10;}.qmmc a, .qmmc li {float:left;display:block;white-space:nowrap;position:relative;z-index:1;}.qmmc div a, .qmmc ul a, .qmmc ul li {float:none;}.qmsh div a {float:left;}.qmmc div{visibility:hidden;position:absolute;}.qmmc .qmcbox{cursor:default;display:block;position:relative;z-index:1;}.qmmc .qmcbox a{display:inline;}.qmmc .qmcbox div{float:none;position:static;visibility:inherit;left:auto;}.qmmc li {z-index:auto;}.qmmc ul {left:-10000px;position:absolute;z-index:10;}.qmmc, .qmmc ul {list-style:none;padding:0px;margin:0px;}.qmmc li a {float:none;}.qmmc li:hover>ul{left:auto;}#qm0 ul {top:100%;}#qm0 ul li:hover>ul{top:0px;left:100%;}
+/*!!!!!!!!!!! QuickMenu Styles [Please Modify!] !!!!!!!!!!!*/
+	/* QuickMenu 0 */
+	/*"""""""" (MAIN) Container""""""""*/
+	#qm0
+	{
+		width:auto;
+		background-color:transparent;
+	}
+	/*"""""""" (MAIN) Items""""""""*/
+	#qm0 a
+	{
+		padding:5px 60px 3px 0px;
+		margin:0px 5px 0px 0px;
+		color:#FFD600;
+		font-family:Arial;
+		font-size:12px;
+		text-decoration:none;
+		font-weight:bold;
+		border-width:0px 0px 8px 0px;
+		border-style:solid;
+		border-color:#FFD600;
+	}
+	/*"""""""" (MAIN) Hover State""""""""*/
+	#qm0 a:hover
+	{
+		color:#3C13AF;
+		border-color:#FFAD00;
+	}
+	/*"""""""" (MAIN) Hover State - (duplicated for pure CSS)""""""""*/
+	#qm0 li:hover>a
+	{
+		color:#3C13AF;
+		border-color:#FFAD00;
+	}
+	/*"""""""" (MAIN) Parent Items""""""""*/
+	#qm0 .qmparent
+	{
+		background-repeat:no-repeat;
+		background-position:95% 50%;
+	}
+	/*"""""""" (MAIN) Active State""""""""*/
+	body #qm0 .qmactive, body #qm0 .qmactive:hover
+	{
+		color:#3C13AF;
+		text-decoration:none;
+		border-color:#FFAD00;
+	}
+	/*"""""""" (SUB) Container""""""""*/
+	#qm0 div, #qm0 ul
+	{
+		padding:10px 5px 10px 5px;
+		background-color:#EEEEEE;
+		border-width:1px;
+		border-style:solid;
+		border-color:#3C13AF;
+	}
+	/*"""""""" (SUB) Items""""""""*/
+	#qm0 div a, #qm0 ul a
+	{
+		padding:3px 5px 3px 5px;
+		background-color:transparent;
+		color:#230672;
+		font-size:11px;
+		font-weight:normal;
+		border-width:0px;
+		border-style:none;
+		border-color:#230672;
+	}
+	/*"""""""" (SUB) Hover State""""""""*/
+	#qm0 div a:hover
+	{
+		background-color:#476DD5;
+		color:#FFFFFF;
+		text-decoration:none;
+	}
+	/*"""""""" (SUB) Hover State - (duplicated for pure CSS)""""""""*/
+	#qm0 ul li:hover>a
+	{
+		background-color:#476DD5;
+		color:#FFFFFF;
+		text-decoration:none;
+	}
+	/*"""""""" (SUB) Parent Items""""""""*/
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