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Latest revision as of 03:34, 22 October 2009

Histamine Sensor Weekly Lab Log

Week 6

July 20, 09

Plan for the week:

Run gels: digest OmpC, TetA
gel purify
ligate Tet into pGEMT-easy→ DH5α transformation
ligate OmpC-TetA standard assembly→ DH5α transformation
OmpR BB: grow cultures tonight→ genomic purification tomorrow
PCR Taz1BB
Amp-Kan-Tet plates
Mutagenic PCR

Tasks accomplished today:

1) Ashley did PCR of Taz1

BioBrick Primers: Tar BB For (19.3 nm); EnvZ Rev (17.1 nm)
Control Primers: Amp Taz 1 For (23.0 nm); Control Taz1 Rev (26.9 nm)

(i) Resuspend dry primers to 100 μM stock (ii) Make 20 μM working stock ( 1μL 100 μM stock + 4 μL H2O= 5 μL total) (iii) Template: Taz1 plasmid (miniprep)

Mastermix: 47 μL
Primer For + Rev: 1 μL each
3 tubes of BioBrick, 3 tubes of control

PCR program:

1) 94°C for 5 min 2) 94°C for 30 sec 3) 57°C* for 30 sec 4) 72°C for 1.5 min 5) GoTo 2, 34 times 6) 72°C for 5 min 7) 4°C forever

2) Gel results for digests:

1% gel

- L1: 1 kb ladder
- L 2: Tet Steph (EcoRI, XbaI)
- L3: Tet MC ( E,X)
- L4: Tet Ash (E,X)
- L5: Tet Steph (E,P)
- L6: Tet MC (E,P)
- L7: Tet Ash (E,P)
- L8: Tet old (E,P)

2% gel

- L1: 1 kb
- L2: Omp2 MC
- L3: Omp1 Steph
- L4 : Omp 2 Steph
- L5: 100 bp ladder
- L6: 100 bp ladder

Did gel extraction on all samples

3) Making competent RU1012

(i) Plated RU1012 agar stab on LB-Kan plate (ii) 11 am: started liquid culture (iii) 6 pm: inoculate into SOB broth (2 mL, 4 mL, and 10 mL samples) (iv) check OD600 with 3 μL sample on nanodrop

11:40 am:
- 2 mL: 0.356
- 4 mL: 0.380
- 10 mL; 0.388

4) Testing transformation efficiency of DH5α (i) add 50 μL competent cells + 1 μL plasmid (OmpR miniprep 7a) (ii) incubate 30 mins on ice (iii) heat shock 30 sec at 42 °C (iv) incubate on ice for 2 min (v) 200 μL LB media (vi) incubate 20 min at 37°C (vii) plate different densities: 100 μL, 10 μL, 1 μL, incubate at 37°C at 4:30 pm (viii) calculate CFU

5) Gel for Taz1

Lane 1: 1 kb ladder
Lane 2: 100 bp ladder
Lane 3: Taz 1
Lane 4: Taz 2

July 21, 09

Tasks for today:

Run digest again, test E and S on tet
Genomic purification of DH5α then PCR OmpR, Run gel
PCR Taz1, Run gels
Calculate competency of gels

1) Digest of Tet (from 7/16 minipreps) with EcoRI and SpeI

Concentration (ng/μL ), μL DNA, μL water
- Tet Steph: 129.3, 7.737, 8.27
- Tet Ash: 126.1, 7.93, 8.07
- Tet Michael: 71.8, 13.93, 2.07

Incubate till 12:00 pm (1.5 hours)

Lane1: 1 kb ladder
Lane 2: Ash
Lane 3: Ash 2
Lane 4: 1 kb ladder
Lane 5: Steph
Lane 6: Steph 2
Lane 7: Michael
Lane 8: 1kb

E, S cut successfully for Tet→ enzymes are ok

2) Genomic purification performed.

Primers: OmpR BB Reverse, OmpR BB Forward
Mastermix: 47 μL; Primers: 1 each, DNA: 1 μL

Lane1: 1 kb ladder
Lane 2: 1 kb ladder
Lane 3: Tube 1
Lane 4: Tube 2
Lane 5: Tube 3
Lane 6: Tube 4
Lane 7: 100 bp ladder
Lane 8: 1kb

Do not use tubes 2 and 3 of genomic DNA

3) PCR of Taz 1

A: amplification. B: Biobrick; Numbers correspond to Taz samples from minipreps of 7/17)

Gel 1
- Lane 1: 1kb
- Lane 2: 1A
- Lane 3: 1B
- Lane 4: 2A
- Lane 5: 2B
- Lane 6: 3A
- Lane 7: 3B
- Lane 8: 1 kb

Gel 2:
- Lane 1: 1kb
- Lane 2: 4A
- Lane 3: 4B
- Lane 4: 5A
- Lane 5: 5B
- Lane 6: 6A
- Lane 7: 6B
- Lane 8: 1 kb

July 22, 09

1) OmpC: Grow liquid cultures, make glycerol stocks, digest with EcoRI and SpeI, run 20 μL on 3% gel with minimal loading dye

2) Sequencing: OmpR, Registry (purified plasmid form), purifying PCR product

3) Sequencing, Registry Taz1→ miniprep transformation in DH5α; make glycerol stocks; purify PCR

Purification of OmpR1, Taz 2B, Taz 4A
Nanodrop concentrations:
- OmpR1: 28.1 ng/μL
- Taz 2B: 16.6 ng/μL
- Taz 4A: 16 ng/μL

Sent in for sequencing

4) tetR: ligation into pGEM T easy (EcoRI, PstI)→ ligation into appropriate expression vector

Digest: 8μL pGEM, 8 μL water, 2 μL multicore buffer, 1 μL EcoRI, 1 μL PstI
Incubate at 37°C

Ligation of TetA into pGEM T-easy
- 10x buffer 2μL
- ligase 1 μL
- vector 1μL
- DNA 10 μL
- Water 6 μL
- Incubate overnight at 4°C

July 23, 09

1) 9am: stop inoculation of ompC, miniprep, nanodrop, sequencing, glycerol stocks 2) Find expression vector for tetR and ligate (from pGEM T-easy-tetR ligation) 3) Design primers for ompC 4) Make RU1012 competent

Taking concentrations of RU1012 00 λ600

10:30 am 0.01
11:30 am 0.03
12:30 pm 0.05
1:10 pm 0.09
2:00 pm 0.12
2:40 pm 0.15
3:10 pm 0.2
3:50 pm 0.26
4:20 pm 0.33
4:50 pm 0.38
5:20 pm 0.50

Completed Inouye protocol, stored at -80°C

July 24, 09

1) Inoculated tet-pGEM T-easy in liquid cultures→ miniprep, nanodrop, digest→ expression vector 2) Run gel:

- Omp: cut with S, P
- Tet: cut with X,P
- Plasmid: 2079 bp
- Tet: 1191 bp
- OmpC: 108 bp

Supercoiled v.s Linear v.s Circular plasmids
Supercoiled: naturally produced by E.coli (i.e. miniprep) runs faster than linear plasmid
Linear: plasmid fom restriction digest
Circular: covalently closed plasmid (i.e ligation) runs slower than linear plasmid

July 25, 09

1) Standard assembly

- 10 μL Ligation mix

2) Gel Extraction 3) Nanodrop Concentrations:

- OmpC1: 1.8 ng/μL
- OmpC2: 3.9 ng/μL
- Tet3: 12.3 ng/μL
- Tet1: 4.6 ng/μL

4) Digest OmpC with SpeI and PstI

Nanodrop concentration:
- Omp1 MC: 30.8 ng/μL
- Omp2 MC: 31.1 ng/μL

5) Ligate Tet3 into OmpC digest 6) Transformation of OmpC-TetA into DH5α and RU1012

Syzmanski Protocol

50 μL cells and 1 μLDNA→ ice for 30 mins
Heat shock 42°C for 30 sec
2 min on ice
200 μL LB
incubate 20 min at 37°C
plate on Amp plates

July 26, 09

1) Inoculated ompC-tetA ligations @ 9:30 am→ miniprep liquid cultures→ glycerol stocks

Week 7

July 27, 09

1) Digests: RBS (SpeI, PstI), TetA (XbaI, PstI), OmpC (SpeI,PstI), TetA-pGEM (XbaI, PstI) 2) Ligation with Digested Tet from July 24th 3) Transform ligation: RBS-tetA→ inoculate ligation→miniprep, glycerol stocks 4) Ran gel: tet-pGEM (digest with X< P); should see 2 bands at 3000 and 1000 bp→ gel purify tetA 5) Digest: pBluscript (160.7 ng/μL)--? Transform into DH5α→ tet plates

July 29, 09

1) Digest SK with X, P 2) Obtain Tet X,P→ ligate into SK

- sequencing
- ligation with RBS (done with optimized protocol and standard protocol, incubated at 4°C overnight

3) Make amp-tet plates 4) Transform RU1012 with Taz1 (incubation started at 4:40 pm)→ run SDS page gel

August 2, 09

1) IPTG induction of Taz1 from RU1012

- 8:30 pm: started liquid cultures (5 mL LB, 5 μL Amp, colony of RU1012+Taz
- 10:30 am: move 0.5 mL liquid culture to 5 mL new liquid culture
- IPTG volumes: 5 μL, 6 μL, 7.5 μL, 10 μL
- 12:30 pm: spun 1 mL aliquot of 6 μL and 7.5 μL liquid cultures, spun 5 mL aliquot of 5 and 10 μL liquid cultures→ freezer
- 1mL cultures
  - resuspend in 200 μL dH2O
  - take 20 μL of dH2O and move to another tube
  - add 2x sample buffer
  - incubate 5 min at 95 °C, vortex, lyse
  - spin at high speed, load supernatant

Week 8

August 4, 09

1) Tested Mutagenic Primers 2) SDS Page of Taz1→ run for 90 min at 121 V, stain overnight 3) DNA purification of PCR TazMut 4) Ligation of purified PCR product: mut Taz into pGEM

August 5, 09

1) Ligation of OmpR BB and Taz1 BB into pGEM T-easy 2) Transformation into DH5α, plate on Amp plates

- ligation was unsuccessful→ redo

August 6, 09

1) Redo transformations

- Control: weird, clear colonies
- Taz MutC: 2 colonies
- Taz MutE: some clear colonies
- Taz MutD: good plate
- Taz MutA: none
- Taz Mut B: some
- Taz BB: some
- OmpR BB: good plate

2) liquid cultures of Taz B,C,D,E, Taz BB and OmpRBB 3) miniprepped all but TazE (nothing grew) 4) Sent in mutagenic products for sequencing

Week 9

August 10,09

1) Digests a. pGEm-OmpR with E,P→ insert b. pGEM-Taz with E,P-→ insert c. July 11 miniprep samples of OmpC to get BB ector with E,P cut sites→ vector backbone d. Ran a gel→ gel extraction e. Revived glycerol stocks of Taz1 f. Inoculated July 27 Taz1-DH5α transformation colonies in liquid cultures

August 11, 09

1) Round the Horn PCR!

Primers: (i) P (phosphorylated)-For Mut R1 Taz 1+ Rev Mut R1Taz1-P (ii) P-For Mut R2 Tot Taz1+ Rev Mut R2 Tot Taz1-P (iii) For Mut R2 A Taz1+ Rev Mut R2 Tot Taz1-P (iv) P-For Mut R2B Taz1+ Rev Mut R2B Taz1-P (v) ForMut R1 Taz1+ Rev Mut R1 Taz1 (linear) (vi) For Mut R2B Taz1+ Rev Mut R 2B Taz1 (linear)

Primer Phosphorylation: (i) 37 μL H2O (ii) 5 μL PNK buffer (iii) 1 μL 50mM MgSO4 (iv) 5 μL primers (100 μM) (v) incubate at 37°C (vi) kill PNK- heat mixture at 95°C for 5 min

August 12, 09

1) started PCR

PCR mixture

39 μL dH2O
5 μL 10x polymerase buffer
1.5 μL forward primer
1.5 μL backward primer
1 μL DNTPs
1 μL template=miniprep Taz1
1 μL Pfx platinum polymerase

PCR program (i) 95°C 1 min (ii) 93 °C 30 sec (iii) 48 °C 30 sec (iv) 72 °C 18 sec (v) Go to 2 25 times (vi) 4 °C hold

2) Loren Looger’s suggestions for changing Tar to become histidine receptor

R69E, R69D, R69Q, R69N
R73E, R73D, R73Q, R73N

August 13, 09

Ligation of PCR products

August 14, 09

1) PCR amplification of Taz with new primers 2) Gel visualization (bright bands!) 3) Gel extraction (Qiagen) 4) Ligation

August 15, 09

1) Transformation results: no colonies on all except Taz1 PCR (150 μL ) and Taz Gel (50 μL )→liquid cultures 2) Ligation again with different optimized combinations→ good results

August 16, 09

1) miniprep of Taz-pGEM ligations 2) Transformation of Taz-pGEm ligations

Week 10

August 17, 09

1) PCR Biobrick Taz→ run gel→ extract→ ligation into pGEM

20μL H2O
25 μL mastermix
1 μL template Taz miniprep 5 from 8/11
2 μL forward BB primers
2 μL reverse BB primers

PCR program: (i) 94°C 5 mins (ii) 94 °C 30 sec (iii) 53.5 °C 30 sec (iv) 72 °C 90 sec (v) Repeat 2-5 34x (vi) 72 °C 5 mins (vii) Hold 4 °C

Nanodrop concentrations of gel extractions of Taz BB PCR:
- Taz BB PCR1: 57.2 ng/μL
- Taz BB PCR2: 66.7 ng/μL

2) Digest Taz 1 minipreps (pGEM) wih Bam and Eco→ gel extract

Taz 1 miniprep (8/16 samples): Nanodrop concentrations (ng/μL)

(1) :95.70 (2): 89.64 (3): 164.52 (4): 150.93 (5): 198.37 (6): 76.46 (7): 86.61 (8): 99.79 Digest at 37°C for two hours. 11:40 am-1:40 pm

3) Ligate Taz 1 into pGEm

Use Team 1’s gel extraction of pGem backbone(17.5 ng/μL), digested with B, E
Ligate digested Taz1 into pGem:
- 3 μL insert, 1 μL vector
- 1 μL ligation buffer (10x)
- 1 μL ligase

Use optimized gel extraction protocol: load 2 20 μL samples in separate wells
Cut as close as possible for each band→ combine in 1 tube
QiaQuick Protocol notes: no unnecessary steps

4) Liquid culture at 5 pm of Taz1-pGem ligations 5) Geneart order

August 18, 09

1) Miniprep of Taz-pGEM 2) Run gel of digest of Bam-Eco on Taz-pGEM→gel extract→ ligation into pNoTat

Lane 1: 1 kb ladder
Lane 2: digest #1
Lane 3: #2
Lane 4: -
Lane 5: #3
Lane 6: #4 (mixture wasn’t 20 μL)
Lane 7: -
Lane 8: #5

Lane 1: 1 kb ladder
Lane 2: -
Lane 3: #6
Lane 4: #7
Lane 5: #8
Lane 6: -
Lane 7: 1 kb ladder
Lane 8: -

Refer to Taz Bam Eco Digest 8/18.tif for images
Digests 1,2,3,6,7,8, came out nicely

Gel extraction:
- Mass (g), Mass+ gel (g), Gel (g), QG buffer:
  - (A) Tube 1-2: 1.0201, 1.1442, 0.1241, 372.3
  - (B) Tube 3: 1.0106, 1.0767, 0.0661, 198.3
  - (C) Tube 6-8: 1.0213, 1.2399, 0.2186, 655.8

Nanodrop concentrations: ng/μL
- A: 8.9
- B: 8.7
- C: 15.9

3) Transformation of Taz BB pGEM 4) Stratagene

August 19, 09

Ligation of TazBB-pGEM (10:30 am -10:45 pm)

1) 6.19 μL H2O 2) 1 μL 10x buffer 3) 1 μL pGEM 4) 1.31 μL Taz 5) 0.5 μL DNA ligase

Transformations into RU 1012

1) Taz pNoTat (4)+C 2) TazBBpGEM (4)+C

August 20, 09

Liquid culture: Testing RU1012 (Kanr) with Taz 1 (Ampr)
Negative: no growth
Amp: no growth
Amp+Kan: growth
Kan: growth
Chloremphenicol: no growth
A+K+C: no growth

Week 10

1. 8/24/09: Received successful sequencing for Taz1 (p-GEM). 2. Received GINKGO ompC-RFP (Tet):

Incubated plates, inoculated cultures, mini-prepped DNA

3. Constructed Taz1 BB:

Digest (EcoR1, Pst1) and gel extraction of mini-prepped Taz1 BB (p-GEM)

Taz1 BB (p-GEM) Mini-Preps (Nanodrop Concentrations): Sample: Concentration, 260/280
- 1: 286.0, 1.93
- 2: 127.3, 1.98
- 3: 330.1, 1.92
- 4: 292.3, 1.95
- 5: 99.6, 1.98
- 6: 415.1, 1.92
- 7: 110.5, 1.94

Gel: Taz1 BB (p-GEM):

Gel Extraction Concentration: Taz1 BB (p-GEM): Sample: ng/uL, 260/280
- 1: 9.8, 1.48
- 2: 13.7, 1.78

Digest (EcoR1, Pst1) and gel extraction of BB vector.

Gel: BB Vector:

Gel Extraction Concentration: BB Vector
- 18.1 ng/uL, 260/280 = 1.83

Overnight ligation of Taz1 BB and BB vector.

For 50 ng BB vector (2.78 uL of 18.1 ng/uL BB vector Gel Extract) (1458/2079) (3 or 6) = 105.19ng (10.73 uL of 9.8ng/uL Taz1 BB (p-GEM) Sample 1 Gel Extract) or 210.39 ng (21.47uL) Taz1 BB.

Transformation of ligation into DH5α. Incubated plates, inoculated liquid cultures, mini-prepped DNA, received successful sequencing.

4. Stratagene Mutagenesis:

(i) Thaw dNTP mix once; prepare single-use aliquots (-20°C) (ii) Control Reaction

5 μL 10 x rxn buffer; 2 μL pwhitescript control plasmid (4.5 kb); 1.25 μL control primers (1), (2); 1 μL dNTP mix; 38.5 μL ddH2O
After: 1 μL pFuUltra DNA polymerase

Reaction:
- 5 μL 20x buffer; DNA template (Taz pNoTat); primer (1 mut), primer (2 mut); 1 μL dNTP; X μL ddH2O
- After: 1 μL PfuUltra DNA polymerase

Transformation→ plate: no colonies for both control snad samples

5. Tested Sequencing of Taz1 (p-NOTat ):

Digest (BamH1, EcoR1) and received successful sequencing.

Taz1 (p-NOTat) Mini-Preps (Nanodrop Concentrations): Sample: Concentration, 260/280
- 1: 178.4, 1.97
- 2: 188.6, 1.96
- 3: 168.7, 1.96
- 4: 371.2, 1.91
- 5: 197.2, 1.95
- 6: 137.5, 2.00
- 7: 198.4, 1.97

Week 12

September 10, 09

(i) control:

5 μL 10x reaction buffer
2 μL pwhitescript
1.25 μL control primer 1
1.25 μL control primer 2
1 μL dNTP
38.5 μL ddH2O
- then 1 μL PfuUltra HF DNA polymerase

(ii) sample (same except 2 μL pNoTat template 6 (137.5 ng/μL)

Result: no colonies

September 13, 09

Transformation of RU1012
T1: RU1012+ OmpC-RFP→ Tet plate (3:20 pm)
T2: RU1012+ OmpC-RFP→ Tet plate (3:20 pm)
T3: RU1012+ OmpC-RFP + Taz→ Tet-Amp plate (3:50 pm)
T4: RU1012+ OmpC-RFP+ Taz→ Tet=Amp Plate (3:50 pm)
T5: RU1012→ Tet plate (3:20 pm)

Week 13

September 14, 09, 7 am

Check plates: RU1012 negative control→ lots of growth!!! Batch of plates is bad.
- (200 μL) RU1012+OmpC-RFP→ lots of growth
- (50 μL) RU1012+ OmpC-RFP→ lots of growth
- MC IPTG+ Xgal→ no growth
- Sample 1 250 μL → no growth
- Sample 2 250 μL→ 3 colonies
- TC IPTG+ X-gal→ 6-7 colonies
- RU1012+Taz+Ompc-RFP 1→ no growth
- RU1012+ Taz+ OmpC-RFP 2→ growth

September 15 -16, 09

Testing Aspartate binding of Double transformants
8:15 pm: start 10 liquid cultures in minimal media with Amp, Tet, Kan
10:15 am: move 0.5 mL to new 5 mL culture tube (minimal media+ A, T,K)

Liquid cultures didn’t grow
Redid cultures→ 16 of them

(i) LB→ growth (ii) LB+A→ growth (iii) LB+T→growth (iv) LB+A+T→ no growth (v) MM→ no growth (vi) MM+A→ no growth (vii) MM+T→ no growth (viii) MM+A+T→ no growth

Plates (LB amp, tet-new plates)
IPTG induced: re-streak

IPTG induced RU1012 Double transformation
- OmpC-RFP (Tetr)
- Taz pNotat miniprep1 from 8/21/09(Ampr)

Redid double transformation of RU1012 with Taz-pNoTat+OmpC-RFP
2:30 pm: visualized under fluroscope→ WE SEE RED!! DOUBLE TRANSFORMATION SUCCESS.

Liquid cultures (picked only red colonies) 5 μL
- LB (negative)→ growth
- LB→ growth
- LB+A→ growth
- LB+T→ no growth
- LB+A+T→ no growth
- MM (neg)→ no growth
- MM→ no growth
- MM+A→ no growth
- MM+T→ no growth
- MM+A+T→ no growth

September 18, 09

To do: try liquid cultures with less Tet
Single transformation of RFP (TetR) Construct→RU1012+ DH5α
Plate on only Tet plate (make sure to use the TetR construct is usd and not the first KanR construct we received)

Week 14

September 21, 09

Retry liquid cultures from LB-A-T plate

Amp Tet Growth
- - Yes
- 0.1 μL Yes
- 0.5 μL Yes
- 1 μL Yes
- 2 μL Yes
- 4 μL Yes
5 μL 0.1 μL Yes
5 μL 0.5 μL Yes
5 μL 1 μL Yes
5 μL 2 μL Yes
5 μL 4 μL Yes

IPTG induction (5 μL) at 10:25 am
- 0.5 mL liquid culture
- 5 mL Minimal media
- 5 μL IPTG

Result: All fluoresced red…there is lactose in LB.

September 23, 09

Minimal media liquid cultures (1pm→ 10 am)
- MM (neg)→ no growth
- MM (pos)+ RU1012 with Taz 1 from 8/20→ growth
- MM with double transformants→ growth
- MM+A with double transformants→ no growth
- MM+T with double transformants→ no growth
- MM+A+T with double transformants→ no growth

Testing for RFP (5mL cultures)

1) no IPTG no aspartate 2) no IPTG 100 μL 0.1M Asp (2mM) 3) 5 μL IPTG 50 μL Asp (1mM) 4) 5 μL IPTG 100 μL Asp (2mM) 5) 5 μL IPTG 10 μL Asp (0.2 mM) 6) 5 μL IPTG (3 hours later)→ 10 μL Asp 7) 5 μL IPTG (3 hours later)→ 50 μL Asp 8) 5 μL IPTG (3 hours later)→ 100 μL Asp

0.1 M Aspartate solution: 1.33 g in 100mL dH2O

@@ Line 51: / Line 51: @@
 ) Gel results for digests:
-% gel
+*1% gel
-L1: 1 kb ladder
+**L1: 1 kb ladder
-L 2: Tet Steph (EcoRI, XbaI)
+**L 2: Tet Steph (EcoRI, XbaI)
-L3: Tet MC ( E,X)
+**L3: Tet MC ( E,X)
-L4: Tet Ash (E,X)
+**L4: Tet Ash (E,X)
-L5: Tet Steph (E,P)
+**L5: Tet Steph (E,P)
-L6: Tet MC (E,P)
+**L6: Tet MC (E,P)
-L7: Tet Ash (E,P)
+**L7: Tet Ash (E,P)
-L8: Tet old (E,P)
+**L8: Tet old (E,P)
-<PHOTO TO GO HERE>
-% gel
+*2% gel
+**L1: 1 kb
+**L2: Omp2 MC
+**L3: Omp1 Steph
+**L4 : Omp 2 Steph
+**L5: 100 bp ladder
+**L6: 100 bp ladder
-L1: 1 kb
-L2: Omp2 MC
-L3: Omp1 Steph
-L4 : Omp 2 Steph
-L5: 100 bp ladder
-L6: 100 bp ladder
-<PHOTO TO GO HERE>
@@ Line 92: / Line 91: @@
 (iv) check OD600 with 3 μL sample on nanodrop
-:40 am:
+*11:40 am:
-mL: 0.356
+**2 mL: 0.356
-mL: 0.380
+**4 mL: 0.380
-mL; 0.388
+**10 mL; 0.388
 ) Testing transformation efficiency of DH5α
@@ Line 109: / Line 108: @@
 ) Gel for Taz1
-Lane 1: 1 kb ladder
+*Lane 1: 1 kb ladder
-Lane 2: 100 bp ladder
+*Lane 2: 100 bp ladder
-Lane 3: Taz 1
+*Lane 3: Taz 1
-Lane 4: Taz 2
+*Lane 4: Taz 2
-<Insert picture here>
 July 21, 09
@@ Line 126: / Line 125: @@
 ) Digest of Tet (from 7/16 minipreps) with EcoRI and SpeI
-Concentration (ng/μL ), μL DNA, μL water
+*Concentration (ng/μL ), μL DNA, μL water
-Tet Steph: 129.3, 7.737, 8.27
+**Tet Steph: 129.3, 7.737, 8.27
-Tet Ash: 126.1, 7.93, 8.07
+**Tet Ash: 126.1, 7.93, 8.07
-Tet Michael: 71.8, 13.93, 2.07
+**Tet Michael: 71.8, 13.93, 2.07
 Incubate till 12:00 pm (1.5 hours)
-<Insert gel here>
-Lane1: 1 kb ladder
+*Lane1: 1 kb ladder
-Lane 2: Ash
+*Lane 2: Ash
-Lane 3: Ash 2
+*Lane 3: Ash 2
-Lane 4: 1 kb ladder
+*Lane 4: 1 kb ladder
-Lane 5: Steph
+*Lane 5: Steph
-Lane 6: Steph 2
+*Lane 6: Steph 2
-Lane 7: Michael
+*Lane 7: Michael
-Lane 8: 1kb
+*Lane 8: 1kb
 *E, S cut successfully for Tet→ enzymes are ok
@@ Line 150: / Line 148: @@
 *Mastermix: 47 μL; Primers: 1 each, DNA: 1 μL
-<Insert gel here>
-Lane1: 1 kb ladder
+*Lane1: 1 kb ladder
-Lane 2: 1 kb ladder
+*Lane 2: 1 kb ladder
-Lane 3: Tube 1
+*Lane 3: Tube 1
-Lane 4: Tube 2
+*Lane 4: Tube 2
-Lane 5: Tube 3
+*Lane 5: Tube 3
-Lane 6: Tube 4
+*Lane 6: Tube 4
-Lane 7: 100 bp ladder
+*Lane 7: 100 bp ladder
-Lane 8: 1kb
+*Lane 8: 1kb
 *Do not use tubes 2 and 3 of genomic DNA
@@ Line 165: / Line 162: @@
 ) PCR of Taz 1
-<Insert gel pictures here>
-A: amplification. B: Biobrick; Numbers correspond to Taz samples from minipreps of 7/17)
+*A: amplification. B: Biobrick; Numbers correspond to Taz samples from minipreps of 7/17)
-Gel 1
+*Gel 1
-Lane 1: 1kb
+**Lane 1: 1kb
-Lane 2: 1A
+**Lane 2: 1A
-Lane 3: 1B
+**Lane 3: 1B
-Lane 4: 2A
+**Lane 4: 2A
-Lane 5: 2B
+**Lane 5: 2B
-Lane 6: 3A
+**Lane 6: 3A
-Lane 7: 3B
+**Lane 7: 3B
-Lane 8: 1 kb
+**Lane 8: 1 kb
-Gel 2:
+*Gel 2:
-Lane 1: 1kb
+**Lane 1: 1kb
-Lane 2: 4A
+**Lane 2: 4A
-Lane 3: 4B
+**Lane 3: 4B
-Lane 4: 5A
+**Lane 4: 5A
-Lane 5: 5B
+**Lane 5: 5B
-Lane 6: 6A
+**Lane 6: 6A
-Lane 7: 6B
+**Lane 7: 6B
-Lane 8: 1 kb
+**Lane 8: 1 kb
 July 22, 09
@@ Line 213: / Line 209: @@
 **10x buffer 2μL
 **ligase 1 μL
-*vector 1μL
+**vector 1μL
 **DNA  10 μL
-	Water 6 μL
+**Water 6 μL
-	Incubate overnight at 4°C
+**Incubate overnight at 4°C
 July 23, 09
@@ Line 326: / Line 322: @@
-----
 Week 8
 ----
@@ Line 498: / Line 494: @@
 ) Run gel of digest of Bam-Eco on Taz-pGEM→gel extract→ ligation into pNoTat
-<Gel picture of Taz 1-pGEM digest (E+B)>
 *Lane 1: 1 kb ladder
@@ Line 587: / Line 582: @@
-Photograph: Gel: Taz1 BB (p-GEM):
+Gel: Taz1 BB (p-GEM):
-<PHOTO TO GO HERE>
@@ Line 599: / Line 592: @@
 *Digest  (EcoR1, Pst1) and gel extraction of BB vector.
-Photograph: Gel: BB Vector:
+Gel: BB Vector:
-<PHOTO TO GO HERE>
@@ Line 650: / Line 642: @@
 September 10, 09
-(i)	control:
+(i) control:
-μL 10x reaction buffer
+*5 μL 10x reaction buffer
-μL pwhitescript
+*2 μL pwhitescript
-.25 μL control primer 1
+*1.25 μL control primer 1
-.25 μL control primer 2
+*1.25 μL control primer 2
-μL dNTP
+*1 μL dNTP
-.5 μL ddH2O
+*38.5 μL ddH2O
-→ then 1 μL PfuUltra HF DNA polymerase
+**then 1 μL PfuUltra HF DNA polymerase
-(ii)	sample (same except 2 μL pNoTat template 6 (137.5 ng/μL)
+(ii) sample (same except 2 μL pNoTat template 6 (137.5 ng/μL)
+*Result: no colonies
+September 13, 09
+*Transformation of RU1012
+*T1: RU1012+ OmpC-RFP→ Tet plate (3:20 pm)
+*T2: RU1012+ OmpC-RFP→ Tet plate (3:20 pm)
+*T3: RU1012+ OmpC-RFP + Taz→ Tet-Amp plate (3:50 pm)
+*T4: RU1012+ OmpC-RFP+ Taz→ Tet=Amp Plate (3:50 pm)
+*T5: RU1012→ Tet plate (3:20 pm)
-Result: no colonies
-Sept 13, 09
-	Transformation of RU1012
-	T1: RU1012+ OmpC-RFP→ Tet plate (3:20 pm)
-	T2: RU1012+ OmpC-RFP→ Tet plate (3:20 pm)
-	T3: RU1012+ OmpC-RFP + Taz→ Tet-Amp plate (3:50 pm)
-	T4: RU1012+ OmpC-RFP+ Taz→ Tet=Amp Plate (3:50 pm)
-	T5: RU1012→ Tet plate (3:20 pm)
-----
 Week 13
 ----
-Sept 14, 09 7 am
+September 14, 09, 7 am
-	Check plates: RU1012 negative control→ lots of growth!!! Batch of plates is bad.
-	(200 μL) RU1012+OmpC-RFP→ lots of growth
-	(50 μL) RU1012+ OmpC-RFP→ lots of growth
-	MC IPTG+ Xgal→ no growth
-	Sample 1 250 μL → no growth
-	Sample 2 250 μL→ 3 colonies
-	TC IPTG+ X-gal→ 6-7 colonies
-	RU1012+Taz+Ompc-RFP 1→ no growth
-	RU1012+ Taz+ OmpC-RFP 2→ growth
-Sept 15+ 16, 09
+*Check plates: RU1012 negative control→ lots of growth!!! Batch of plates is bad.
-	- Testing Aspartate binding of Double transformants
+**(200 μL) RU1012+OmpC-RFP→ lots of growth
-:15 pm: start 10 liquid cultures in minimal media with Amp, Tet, Kan
+**(50 μL) RU1012+ OmpC-RFP→ lots of growth
-:15 am: move 0.5 mL to new 5 mL culture tube (minimal media+ A, T,K)
+**MC IPTG+ Xgal→ no growth
+**Sample 1 250 μL → no growth
+**Sample 2 250 μL→ 3 colonies
+**TC IPTG+ X-gal→ 6-7 colonies
+**RU1012+Taz+Ompc-RFP 1→ no growth
+**RU1012+ Taz+ OmpC-RFP 2→ growth
+September 15 -16, 09
+*Testing Aspartate binding of Double transformants
+*8:15 pm: start 10 liquid cultures in minimal media with Amp, Tet, Kan
+*10:15 am: move 0.5 mL to new 5 mL culture tube (minimal media+ A, T,K)
-	- Liquid cultures didn’t grow
+*Liquid cultures didn’t grow
-	Redid cultures→ 16 of them
+*Redid cultures→ 16 of them
-(i)	LB→ growth
-(ii)	LB+A→ growth
+(i) LB→ growth
-(iii)	LB+T→growth
+(ii) LB+A→ growth
-(iv)	LB+A+T→ no growth
+(iii) LB+T→growth
-(v)	MM→ no growth
+(iv) LB+A+T→ no growth
-(vi)	MM+A→ no growth
+(v) MM→ no growth
+(vi) MM+A→ no growth
 (vii)	MM+T→ no growth
 (viii)	MM+A+T→ no growth
-Plates (LB amp, tet-new plates)
+*Plates (LB amp, tet-new plates)
-IPTG induced: re-streak
+*IPTG induced: re-streak
--	IPTG induced RU1012 Double transformation
+*IPTG induced RU1012 Double transformation
-o	OmpC-RFP (Tetr)
+**OmpC-RFP (Tetr)
-o	Taz pNotat miniprep1 from 8/21/09(Ampr)
+**Taz pNotat miniprep1 from 8/21/09(Ampr)
-Redid double transformation of RU1012 with Taz-pNoTat+OmpC-RFP
+*Redid double transformation of RU1012 with Taz-pNoTat+OmpC-RFP
-:30 pm: visualized under fluroscope→ WE SEE RED!! DOUBLE TRANSFORMATION SUCCESS.
+*2:30 pm: visualized under fluroscope→ WE SEE RED!! DOUBLE TRANSFORMATION SUCCESS.
-Liquid cultures (picked only red colonies) 5 μL
+*Liquid cultures (picked only red colonies) 5 μL
-LB (negative)→ growth
+**LB (negative)→ growth
-LB→ growth
+**LB→ growth
-LB+A→ growth
+**LB+A→ growth
-LB+T→ no growth
+**LB+T→ no growth
-LB+A+T→ no growth
+**LB+A+T→ no growth
-MM (neg)→ no growth
+**MM (neg)→ no growth
-MM→ no growth
+**MM→ no growth
-MM+A→ no growth
+**MM+A→ no growth
-MM+T→ no growth
+**MM+T→ no growth
-MM+A+T→ no growth
+**MM+A+T→ no growth
+September 18, 09
+*To do: try liquid cultures with less Tet
+*Single transformation of RFP (TetR) Construct→RU1012+ DH5α
+*Plate on only Tet plate (make sure to use the TetR construct is usd and not the first KanR construct we received)
-Sept 18, 09
-	To do: try liquid cultures with less Tet
-	Single transformation of RFP (TetR) Construct→RU1012+ DH5α
-	Plate on only Tet plate (make sure to use the TetR construct is usd and not the first KanR
-             construct we received)
-----
 Week 14
 ----
@@ Line 740: / Line 739: @@
-Sept 21, 09
+September 21, 09
-	Retry liquid cultures from LB-A-T plate
-Amp	Tet	Growth
+*Retry liquid cultures from LB-A-T plate
--	-	Yes
--	0.1 μL	Yes
+*Amp	Tet	Growth
--	0.5 μL	Yes
+*-	-	Yes
--	1 μL	Yes
+*-	0.1 μL   Yes
-- 	2 μL	Yes
+*-	0.5 μL   Yes
-- 	4 μL	Yes
+*-	1 μL	   Yes
-μL	0.1 μL	Yes
+*- 	2 μL   Yes
-μL	0.5 μL	Yes
+*- 	4 μL   Yes
-μL	1 μL	Yes
+*5 μL   0.1 μL   Yes
-μL	2 μL	Yes
+*5 μL   0.5 μL   Yes
-μL	4 μL	Yes
+*5 μL   1 μL   Yes
+*5 μL   2 μL   Yes
+*5 μL   4 μL   Yes
+*IPTG induction (5 μL) at 10:25 am
+**0.5 mL liquid culture
+**5 mL Minimal media
+**5 μL IPTG
+*Result: All fluoresced red…there is lactose in LB.
-IPTG induction (5 μL) at 10:25 am
-.5 mL liquid culture
-mL Minimal media
-μL IPTG
-Result: All fluoresced red…there is lactose in LB.
+September 23, 09
-Sept 23, 09
+*Minimal media liquid cultures (1pm→ 10 am)
-	Minimal media liquid cultures (1pm→ 10 am)
+**MM (neg)→ no growth
-	MM (neg)→ no growth
+**MM (pos)+ RU1012 with Taz 1 from 8/20→ growth
-	MM (pos)+ RU1012 with Taz 1 from 8/20→ growth
+**MM with double transformants→ growth
-	MM with double transformants→ growth
+**MM+A with double transformants→ no growth
-	MM+A with double transformants→ no growth
+**MM+T with double transformants→ no growth
-	MM+T with double transformants→ no growth
+**MM+A+T with double transformants→ no growth
-	MM+A+T with double transformants→ no growth
-	Testing for RFP (5mL cultures)
+*Testing for RFP (5mL cultures)
-)	no IPTG no aspartate
+) no IPTG no aspartate
-)	no IPTG 100 μL 0.1M Asp (2mM)
+) no IPTG 100 μL 0.1M Asp (2mM)
-)	5 μL IPTG 50 μL Asp (1mM)
+) 5 μL IPTG 50 μL Asp (1mM)
-)	5 μL IPTG 100 μL Asp (2mM)
+) 5 μL IPTG 100 μL Asp (2mM)
-)	5 μL IPTG 10 μL Asp (0.2 mM)
+) 5 μL IPTG 10 μL Asp (0.2 mM)
-)	5 μL IPTG (3 hours later)→ 10 μL Asp
+) 5 μL IPTG (3 hours later)→ 10 μL Asp
-)	5 μL IPTG (3 hours later)→ 50 μL Asp
+) 5 μL IPTG (3 hours later)→ 50 μL Asp
-)	5 μL IPTG (3 hours later)→ 100 μL Asp
+) 5 μL IPTG (3 hours later)→ 100 μL Asp
-.1 M Aspartate solution: 1.33 g in 100mL dH2O
+*0.1 M Aspartate solution: 1.33 g in 100mL dH2O

Team:Brown/Notebook weekly Logs/Weekly Team2 Notebook

From 2009.igem.org

Latest revision as of 03:34, 22 October 2009

Histamine Sensor Weekly Lab Log