Team:Lethbridge/Notebook

From 2009.igem.org

(Difference between revisions)
(→July)
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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.  
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.
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Revision as of 22:20, 17 September 2009


Igembanner.jpg



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.

August

September

October

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