Team:Brown/Notebook weekly Logs/Weekly Team2 Notebook

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(New page: <div style="background-color:black"> {{Brown}} <font><font color="white"> Team 2 Histamine Sensor Weekly Lab Log ---- ---- Week 6 ---- Jul 20, 09 Plan for the week: • Run...)
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Team 2 Histamine Sensor Weekly Lab Log
+
=Histamine Sensor Weekly Lab Log=
-
----
 
-
----
 
Week 6
Week 6
----
----
-
Jul 20, 09
+
July 20, 09
Plan for the week:
Plan for the week:
-
• Run gels: digest OmpC, TetA
+
*Run gels: digest OmpC, TetA
-
        → gel purify
+
*gel purify
-
→ ligate Tet into pGEMT-easy→ DH5α transformation
+
*ligate Tet into pGEMT-easy→ DH5α transformation
-
→ ligate OmpC-TetA standard assembly→ DH5α transformation
+
*ligate OmpC-TetA standard assembly→ DH5α transformation
-
• OmpR BB: grow cultures tonight→ genomic purification tomorrow
+
*OmpR BB: grow cultures tonight→ genomic purification tomorrow
-
• PCR Taz1BB
+
*PCR Taz1BB
-
• Amp-Kan-Tet plates
+
*Amp-Kan-Tet plates
-
• Mutagenic PCR
+
*Mutagenic PCR
Tasks accomplished today:
Tasks accomplished today:
1) Ashley did PCR of Taz1
1) Ashley did PCR of Taz1
-
-BioBrick Primers: Tar BB For (19.3 nm); EnvZ  Rev (17.1 nm)
+
*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)
+
*Control Primers:  Amp Taz 1 For  (23.0 nm); Control Taz1 Rev (26.9 nm)
-
(i) Resuspend dry primers to 100 μM stock
+
(i) Resuspend dry primers to 100 μM stock
-
(ii) Make 20 μM working stock ( 1μL 100 μM stock + 4 μL H2O= 5 μL total)
+
(ii) Make 20 μM working stock ( 1μL 100 μM stock + 4 μL H2O= 5 μL total)
-
(iii) Template: Taz1 plasmid (miniprep)
+
(iii) Template: Taz1 plasmid (miniprep)
-
Mastermix: 47 μL
+
 
-
Primer For + Rev: 1 μL each
+
*Mastermix: 47 μL
-
3 tubes of BioBrick, 3 tubes of control
+
*Primer For + Rev: 1 μL each
-
PCR program:  
+
*3 tubes of BioBrick, 3 tubes of control
 +
 
 +
*PCR program:  
1) 94°C for 5 min
1) 94°C for 5 min
2) 94°C for 30 sec
2) 94°C for 30 sec
Line 54: Line 49:
7) 4°C forever
7) 4°C forever
-
2) Gel results for digests:
+
2) Gel results for digests:  
 +
 
1% gel
1% gel
 +
L1: 1 kb ladder
L1: 1 kb ladder
L 2: Tet Steph (EcoRI, XbaI)
L 2: Tet Steph (EcoRI, XbaI)
Line 86: Line 83:
-
• Did gel extraction on all samples
+
*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
-
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:  
11:40 am:  
-
2 mL: 0.356
+
2 mL: 0.356
-
4 mL: 0.380
+
4 mL: 0.380
-
10 mL; 0.388
+
10 mL; 0.388
-
4) Testing transformation efficiency of DH5α
+
 
-
(i) add 50 μL competent cells + 1 μL  plasmid (OmpR miniprep 7a)
+
4) Testing transformation efficiency of DH5α
-
(ii) incubate 30 mins on ice
+
(i) add 50 μL competent cells + 1 μL  plasmid (OmpR miniprep 7a)
-
(iii) heat shock 30 sec at 42 °C  
+
(ii) incubate 30 mins on ice
-
(iv) incubate on ice for 2 min
+
(iii) heat shock 30 sec at 42 °C  
-
(v) 200 μL  LB media
+
(iv) incubate on ice for 2 min
-
(vi) incubate 20 min at 37°C  
+
(v) 200 μL  LB media
-
(vii) plate different densities: 100 μL, 10 μL, 1 μL, incubate at 37°C at 4:30 pm
+
(vi) incubate 20 min at 37°C  
-
(viii) calculate CFU
+
(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
-
5) 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
+
 
 +
<Insert picture here>
July 21, 09
July 21, 09
Tasks for today:  
Tasks for today:  
-
• Run digest again, test E and S on tet
+
*Run digest again, test E and S on tet
-
• Genomic purification of DH5α then PCR OmpR, Run gel
+
*Genomic purification of DH5α then PCR OmpR, Run gel
-
• PCR Taz1, Run gels
+
*PCR Taz1, Run gels
-
• Calculate competency of gels
+
*Calculate competency of gels
-
1) Digest of Tet (from 7/16 minipreps) with EcoRI and SpeI
+
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
+
Concentration (ng/μL ), μL DNA, μL water
-
Tet Ash 126.1 7.93 8.07
+
Tet Steph: 129.3, 7.737, 8.27
-
Tet Michael 71.8 13.93 2.07
+
Tet Ash: 126.1, 7.93, 8.07
 +
Tet Michael: 71.8, 13.93, 2.07
Incubate till 12:00 pm (1.5 hours)
Incubate till 12:00 pm (1.5 hours)
-
Insert gel here
+
 
 +
<Insert gel here>
 +
 
Lane1: 1 kb ladder
Lane1: 1 kb ladder
Lane 2: Ash
Lane 2: Ash
Line 139: Line 144:
Lane 8: 1kb
Lane 8: 1kb
-
E, S cut successfully for Tet→ enzymes are ok
+
*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 
 +
 
 +
<Insert gel here>
-
2) Genomic purification performed.
 
-
Primers: OmpR BB Reverse, OmpR BB Forward
 
-
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
Line 154: Line 161:
Lane 8: 1kb
Lane 8: 1kb
-
→ Do not use tubes 2 and 3 of genomic DNA
+
*Do not use tubes 2 and 3 of genomic DNA
-
3) PCR of Taz 1
+
 
-
Insert gel pictures here
+
3) 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
Line 179: Line 190:
July 22, 09
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
+
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
-
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
-
Purification of OmpR1, Taz 2B, Taz 4A
+
*Sent in for sequencing
-
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
-
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
-
Digest: 8μL pGEM, 8 μL water, 2 μL multicore buffer, 1 μL EcoRI, 1 μL PstI
+
*Ligation of TetA into pGEM T-easy
-
Incubate at 37°C
+
**10x buffer 2μL
-
Ligation of TetA into pGEM T-easy
+
**ligase 1 μL
-
10x buffer 2μL
+
*vector 1μL
-
ligase 1 μL
+
**DNA  10 μL
-
vector 1μL
+
-
DNA  10 μL
+
Water 6 μL
Water 6 μL
Incubate overnight at 4°C
Incubate overnight at 4°C
July 23, 09
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)
+
1) 9am: stop inoculation of ompC, miniprep, nanodrop, sequencing, glycerol stocks
-
3) Design primers for ompC
+
2) Find expression vector for tetR and ligate (from pGEM T-easy-tetR ligation)
-
4) Make RU1012 competent  
+
3) Design primers for ompC
 +
4) Make RU1012 competent  
 +
 
Taking concentrations of RU1012  00 λ600
Taking concentrations of RU1012  00 λ600
-
10:30 am 0.01
+
*10:30 am 0.01
-
11:30 am 0.03
+
*11:30 am 0.03
-
12:30 pm 0.05
+
*12:30 pm 0.05
-
1:10 pm 0.09
+
*1:10 pm 0.09
-
2:00 pm 0.12
+
*2:00 pm 0.12
-
2:40 pm 0.15
+
*2:40 pm 0.15
-
3:10 pm 0.2
+
*3:10 pm 0.2
-
3:50 pm 0.26
+
*3:50 pm 0.26
-
4:20 pm 0.33
+
*4:20 pm 0.33
-
4:50 pm 0.38
+
*4:50 pm 0.38
-
5:20 pm 0.50
+
*5:20 pm 0.50
 +
 
 +
*Completed Inouye protocol, stored at -80°C
-
Completed Inouye protocol, stored at -80°C
 
July 24, 09
July 24, 09
-
1) Inoculated tet-pGEM T-easy in liquid cultures→ miniprep, nanodrop, digest→ expression vector
 
-
2) Run gel:  
+
1) Inoculated tet-pGEM T-easy in liquid cultures→ miniprep, nanodrop, digest→ expression vector
-
Omp: cut with S, P
+
2) Run gel:  
-
Tet: cut with X,P
+
**Omp: cut with S, P
-
Plasmid: 2079 bp
+
**Tet: cut with X,P
-
Tet: 1191 bp
+
**Plasmid: 2079 bp
-
OmpC: 108 bp
+
**Tet: 1191 bp
 +
**OmpC: 108 bp
-
Supercoiled v.s Linear v.s Circular plasmids
+
*Supercoiled v.s Linear v.s Circular plasmids
-
 
+
*Supercoiled: naturally produced by E.coli (i.e. miniprep) runs faster than linear plasmid
-
Supercoiled: naturally produced by E.coli (i.e. miniprep) runs faster than linear plasmid
+
*Linear: plasmid fom restriction digest
-
Linear: plasmid fom restriction digest
+
*Circular: covalently closed plasmid (i.e ligation) runs slower than linear plasmid
-
Circular: covalently closed plasmid (i.e ligation) runs slower than linear plasmid
+
July 25, 09
July 25, 09
-
1) Standard assembly
+
 
-
10 μL Ligation mix
+
1) Standard assembly
-
2) Gel Extraction
+
**10 μL Ligation mix
-
3) Nanodrop Concentrations:
+
 
-
OmpC1: 1.8 ng/μL
+
2) Gel Extraction
-
OmpC2: 3.9 ng/μL
+
3) Nanodrop Concentrations:
-
Tet3: 12.3 ng/μL
+
**OmpC1: 1.8 ng/μL
-
Tet1: 4.6 ng/μL
+
**OmpC2: 3.9 ng/μL
-
4) Digest OmpC with SpeI and PstI
+
**Tet3: 12.3 ng/μL
-
          Nanodrop concentration:
+
**Tet1: 4.6 ng/μL
-
Omp1 MC: 30.8 ng/μL
+
 
-
Omp2 MC: 31.1 ng/μL
+
4) Digest OmpC with SpeI and PstI
-
5) Ligate Tet3 into OmpC digest
+
*Nanodrop concentration:
-
6) Transformation of OmpC-TetA into DH5α and RU1012
+
**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
Syzmanski Protocol
-
- 50 μL cells and 1 μLDNA→ ice for 30 mins
+
*50 μL cells and 1 μLDNA→ ice for 30 mins
-
- Heat shock 42°C for 30 sec
+
*Heat shock 42°C for 30 sec
-
- 2 min on ice
+
*2 min on ice
-
- 200 μL LB
+
*200 μL LB
-
- incubate 20 min at 37°C
+
*incubate 20 min at 37°C
-
- plate on Amp plates
+
*plate on Amp plates
July 26, 09
July 26, 09
-
1) Inoculated ompC-tetA ligations @ 9:30 am→ miniprep liquid cultures→ glycerol stocks
+
 
 +
1) Inoculated ompC-tetA ligations @ 9:30 am→ miniprep liquid cultures→ glycerol stocks
-
----
 
Week 7
Week 7
----
----
July 27, 09
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
+
1) Digests: RBS (SpeI, PstI), TetA (XbaI, PstI), OmpC (SpeI,PstI), TetA-pGEM (XbaI, PstI)
-
3) Transform ligation: RBS-tetA→ inoculate ligation→miniprep, glycerol stocks
+
2) Ligation with Digested Tet from July 24th
-
4) Ran gel: tet-pGEM (digest with X< P); should see 2 bands at 3000 and 1000 bp→ gel purify tetA
+
3) Transform ligation: RBS-tetA→ inoculate ligation→miniprep, glycerol stocks
-
5) Digest: pBluscript (160.7 ng/μL)--? Transform into DH5α→ tet plates
+
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
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
 
-
Aug 2, 09
+
1) Digest SK with X, P
-
1) IPTG induction of Taz1 from RU1012
+
2) Obtain Tet X,P→ ligate into SK
-
8:30 pm: started liquid cultures (5 mL LB, 5 μL Amp, colony of RU1012+Taz
+
**sequencing
-
10:30 am: move 0.5 mL liquid culture to 5 mL new liquid culture
+
**ligation with RBS (done with optimized protocol and standard protocol, incubated at 4°C overnight
-
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
+
3) Make amp-tet plates
-
      spun 5 mL aliquot of 5 and 10 μL liquid cultures→ freezer
+
4) Transform RU1012 with Taz1 (incubation started at 4:40 pm)→ run SDS page gel
-
1mL cultures
+
 
-
-resuspend in 200 μL dH2O
+
August 2, 09
-
- take 20 μL of dH2O and move to another tube
+
 
-
- add 2x sample buffer
+
1) IPTG induction of Taz1 from RU1012
-
- incubate 5 min at 95 °C, vortex, lyse
+
**8:30 pm: started liquid cultures (5 mL LB, 5 μL Amp, colony of RU1012+Taz
-
- spin at high speed, load supernatant
+
**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
Line 308: Line 331:
-
Aug 4, 09  
+
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
+
-
Aug 5, 09
+
1) Tested Mutagenic Primers
-
1) Ligation of OmpR BB and Taz1 BB into pGEM T-easy
+
2) SDS Page of Taz1→ run for 90 min at 121 V, stain overnight
-
2) Transformation into DH5α, plate on Amp plates
+
3) DNA purification of PCR TazMut
-
→ ligation was unsuccessful→ redo
+
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
-
Aug 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
Week 9
----
----
-
Aug 10,09
+
August 10,09
 +
 
1) Digests
1) Digests
a. pGEm-OmpR with E,P→ insert
a. pGEm-OmpR with E,P→ insert
Line 348: Line 376:
f. Inoculated July 27 Taz1-DH5α transformation colonies in liquid cultures
f. Inoculated July 27 Taz1-DH5α transformation colonies in liquid cultures
-
Aug 11, 09
+
August 11, 09
-
1) Round the Horn PCR!
+
 
 +
1) Round the Horn PCR!
 +
 
Primers:
Primers:
-
(i) P (phosphorylated)-For Mut R1 Taz 1+ Rev Mut R1Taz1-P
+
(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
+
(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
+
(iii) For Mut R2 A Taz1+ Rev Mut R2 Tot Taz1-P
-
(iv) P-For Mut R2B Taz1+ Rev Mut R2B Taz1-P
+
(iv) P-For Mut R2B Taz1+ Rev Mut R2B Taz1-P
-
(v) ForMut R1 Taz1+ Rev Mut R1 Taz1 (linear)
+
(v) ForMut R1 Taz1+ Rev Mut R1 Taz1 (linear)
-
(vi) For Mut R2B Taz1+ Rev Mut R 2B Taz1 (linear)
+
(vi) For Mut R2B Taz1+ Rev Mut R 2B Taz1 (linear)
-
                     Primer Phosphorylation
+
                      
-
(i) 37 μL H2O
+
Primer Phosphorylation:
-
(ii) 5 μL PNK buffer
+
(i) 37 μL H2O
-
(iii) 1 μL 50mM MgSO4
+
(ii) 5 μL PNK buffer
-
(iv) 5 μL primers (100 μM)
+
(iii) 1 μL 50mM MgSO4
-
(v) incubate at 37°C
+
(iv) 5 μL primers (100 μM)
-
(vi) kill PNK- heat mixture at 95°C for 5 min
+
(v) incubate at 37°C
 +
(vi) kill PNK- heat mixture at 95°C for 5 min
 +
 
 +
August 12, 09
 +
 
 +
1) started PCR
-
Aug 12, 09
 
-
1) started PCR
 
PCR mixture
PCR mixture
-
39 μL dH2O
+
*39 μL dH2O
-
5 μL 10x polymerase buffer
+
*5 μL 10x polymerase buffer
-
1.5 μL forward primer
+
*1.5 μL forward primer
-
1.5 μL backward primer
+
*1.5 μL backward primer
-
1 μL DNTPs
+
*1 μL DNTPs
-
1 μL template=miniprep Taz1
+
*1 μL template=miniprep Taz1
-
1 μL Pfx platinum polymerase
+
*1 μL Pfx platinum polymerase
PCR program
PCR program
-
(i) 95°C 1 min
+
(i) 95°C 1 min
-
(ii) 93 °C  30 sec
+
(ii) 93 °C  30 sec
-
(iii) 48 °C  30 sec
+
(iii) 48 °C  30 sec
-
(iv) 72 °C  18 sec
+
(iv) 72 °C  18 sec
-
(v) Go to 2 25 times
+
(v) Go to 2 25 times
-
(vi) 4 °C  hold
+
(vi) 4 °C  hold
-
2) Loren Looger’s suggestions for changing Tar to become histidine receptor
+
2) Loren Looger’s suggestions for changing Tar to become histidine receptor
-
R69E, R69D, R69Q, R69N
+
*R69E, R69D, R69Q, R69N
-
R73E, R73D, R73Q, R73N
+
*R73E, R73D, R73Q, R73N
-
Aug 13, 09
+
August 13, 09
-
-Ligation of PCR products
+
-
Aug 14, 09
+
*Ligation of PCR products
-
1) PCR amplification of Taz with new primers
+
-
2) Gel visualization (bright bands!)
+
-
3) Gel extraction (Qiagen)
+
-
4) Ligation
+
-
Aug 15, 09
+
August 14, 09
-
1) Transformation results: no colonies on all except Taz1 PCR (150 μL ) and Taz Gel (50 μL )→
+
 
-
liquid cultures
+
1) PCR amplification of Taz with new primers
-
2) Ligation again with different optimized combinations→ good results
+
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
-
Aug 16, 09
 
-
1) miniprep of Taz-pGEM ligations
 
-
2) Transformation of Taz-pGEm ligations
 
-
----
 
Week 10
Week 10
----
----
-
Aug 17, 09
+
August 17, 09
-
1) PCR Biobrick Taz→ run gel→ extract→ ligation into pGEM
+
 
-
20μL H2O
+
1) PCR Biobrick Taz→ run gel→ extract→ ligation into pGEM
-
25 μL mastermix
+
 
-
1 μL template Taz miniprep 5 from 8/11
+
*20μL H2O
-
2 μL forward BB primers
+
*25 μL mastermix
-
2 μL reverse BB primers
+
*1 μL template Taz miniprep 5 from 8/11
 +
*2 μL forward BB primers
 +
*2 μL reverse BB primers
PCR program:  
PCR program:  
Line 429: Line 467:
(vii) Hold 4 °C  
(vii) Hold 4 °C  
-
Nanodrop concentrations of gel extractions of Taz BB PCR:
+
*Nanodrop concentrations of gel extractions of Taz BB PCR:
-
Taz BB PCR1: 57.2 ng/μL
+
**Taz BB PCR1: 57.2 ng/μL
-
Taz BB PCR2: 66.7 ng/μL
+
**Taz BB PCR2: 66.7 ng/μL
-
2) Digest Taz 1 minipreps (pGEM) wih Bam and Eco→ gel extract
+
2) Digest Taz 1 minipreps (pGEM) wih Bam and Eco→ gel extract
-
Taz 1 miniprep (8/16 samples): Nanodrop concentrations (ng/μL)  
+
*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
(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
Digest at 37°C for two hours. 11:40 am-1:40 pm
-
3) Ligate Taz 1 into pGEm
+
3) Ligate Taz 1 into pGEm
-
Use Team  1’s gel extraction of pGem backbone(17.5 ng/μL), digested with B, E
+
*Use Team  1’s gel extraction of pGem backbone(17.5 ng/μL), digested with B, E
-
Ligate digested Taz1 into pGem:  
+
*Ligate digested Taz1 into pGem:  
-
3 μL insert, 1 μL vector
+
**3 μL insert, 1 μL vector
-
1 μL ligation buffer (10x)
+
**1 μL ligation buffer (10x)
-
1 μL ligase
+
**1 μL ligase
-
Use optimized gel extraction protocol: load 2 20 μL samples in separate wells
+
*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
+
*Cut as close as possible for each band→ combine in 1 tube
-
QiaQuick Protocol notes: no unnecessary steps
+
*QiaQuick Protocol notes: no unnecessary steps
-
4) Liquid culture at 5 pm of Taz1-pGem ligations
+
4) Liquid culture at 5 pm of Taz1-pGem ligations
-
5) Geneart order
+
5) Geneart order
-
Aug 18, 09
 
-
1) Miniprep of Taz-pGEM
 
-
2) Run gel of digest of Bam-Eco on Taz-pGEM→gel extract→ ligation into pNoTat
 
-
Gel picture of Taz 1-pGEM digest (E+B)
+
August 18, 09
-
Lane 1: 1 kb ladder
+
1) Miniprep of Taz-pGEM
-
Lane 2: digest #1
+
2) Run gel of digest of Bam-Eco on Taz-pGEM→gel extract→ ligation into pNoTat
-
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
+
<Gel picture of Taz 1-pGEM digest (E+B)>
-
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
+
*Lane 1: 1 kb ladder
-
Digests 1,2,3,6,7,8, came out nicely
+
*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
-
Gel extraction:
+
*Lane 1: 1 kb ladder
-
Mass (g) Mass+ gel (g) Gel (g) QG buffer
+
*Lane 2: -
-
(A) Tube 1-2 1.0201 1.1442 0.1241 372.3
+
*Lane 3: #6
-
(B) Tube 3 1.0106 1.0767 0.0661 198.3
+
*Lane 4: #7
-
(C) Tube 6-8 1.0213 1.2399 0.2186 655.8
+
*Lane 5: #8
 +
*Lane 6: -
 +
*Lane 7: 1 kb ladder
 +
*Lane 8: -
-
Nanodrop concentrations: ng/μL
+
*Refer to Taz Bam Eco Digest 8/18.tif for images
-
A: 8.9
+
*Digests 1,2,3,6,7,8, came out nicely
-
B: 8.7
+
-
C: 15.9
+
-
3) Transformation of Taz BB pGEM
+
*Gel extraction:
-
4) Stratagene
+
**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
-
Aug 19, 09
+
*Nanodrop concentrations: ng/μL
-
Ligation of TazBB-pGEM (10:30 am -10:45 pm)
+
**A: 8.9
-
1) 6.19 μL H2O
+
**B: 8.7
-
2) 1 μL 10x buffer
+
**C: 15.9
-
3) 1 μL pGEM
+
-
4) 1.31 μL Taz
+
-
5) 0.5 μL DNA ligase
+
-
Transformations into RU 1012
+
3) Transformation of Taz BB pGEM
-
1) Taz pNoTat (4)+C
+
4) Stratagene
-
2) TazBBpGEM (4)+C
+
 
 +
 
 +
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
-
Aug 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
Week 10
----
----
   
   
-
1. 8/24/09: Received successful sequencing for Taz1 (p-GEM).  
+
1. 8/24/09: Received successful sequencing for Taz1 (p-GEM).  
-
2. Received GINKGO ompC-RFP (Tet):  
+
2. Received GINKGO ompC-RFP (Tet):  
-
• Incubated plates, inoculated cultures, mini-prepped DNA
+
-
3. Constructed Taz1 BB: 
+
*Incubated plates, inoculated cultures, mini-prepped DNA
-
• Digest (EcoR1, Pst1) and gel extraction of mini-prepped Taz1 BB (p-GEM)
+
-
Taz1 BB (p-GEM) Mini-Preps (Nanodrop Concentrations):  
+
3. Constructed Taz1 BB: 
-
      Sample                 Concentration         260/280
+
*Digest (EcoR1, Pst1) and gel extraction of mini-prepped Taz1 BB (p-GEM)
-
1 286.0 1.93
+
 
-
2 127.3 1.98
+
*Taz1 BB (p-GEM) Mini-Preps (Nanodrop Concentrations): Sample: Concentration, 260/280
-
3 330.1 1.92
+
**1: 286.0, 1.93
-
4 292.3 1.95
+
**2: 127.3, 1.98
-
5 99.6 1.98
+
**3: 330.1, 1.92
-
6 415.1 1.92
+
**4: 292.3, 1.95
-
7 110.5 1.94
+
**5: 99.6, 1.98
 +
**6: 415.1, 1.92
 +
**7: 110.5, 1.94
-
Photograph: Gel: Taz1 BB (p-GEM):
 
-
 
 +
Photograph: Gel: Taz1 BB (p-GEM):
Line 547: Line 593:
 +
*Gel Extraction Concentration: Taz1 BB (p-GEM): Sample: ng/uL, 260/280
 +
**1: 9.8, 1.48   
 +
**2: 13.7, 1.78  
-
Gel Extraction Concentration: Taz1 BB (p-GEM)
+
*Digest  (EcoR1, Pst1) and gel extraction of BB vector.  
-
Sample ng/uL 260/280
+
-
1 9.8 1.48   
+
-
2 13.7 1.78  
+
-
 
+
-
• Digest  (EcoR1, Pst1) and gel extraction of BB vector.  
+
-
Photograph: Gel: BB Vector:  
+
Photograph: Gel: BB Vector:  
-
 
+
Line 564: Line 607:
-
Gel Extraction Concentration: BB Vector
+
*Gel Extraction Concentration: BB Vector
-
18.1 ng/uL 260/280 = 1.83
+
**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.
+
*Overnight ligation of Taz1 BB and BB vector.  
-
• Transformation of ligation into DH5α. Incubated plates, inoculated liquid cultures, mini-prepped DNA, received successful sequencing.
+
-
4. Stratagene Mutagenesis:
+
*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.  
-
(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:
+
*Transformation of ligation into DH5α. Incubated plates, inoculated liquid cultures, mini-prepped DNA, received successful sequencing.
-
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
+
4. Stratagene Mutagenesis:  
-
5. Tested Sequencing of Taz1 (p-NOTat ):  
+
(i) Thaw dNTP mix once; prepare single-use aliquots (-20°C)
-
• Digest (BamH1, EcoR1) and received successful sequencing.
+
(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
-
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
Week 12
----
----
 +
September 10, 09
-
Sept 10, 09
 
(i) control:  
(i) control:  
5 μL 10x reaction buffer
5 μL 10x reaction buffer

Revision as of 18:36, 21 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)


<PHOTO TO GO HERE>


2% gel

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>


  • 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

<Insert picture here>

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)

<Insert gel here>

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

<Insert gel here>

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

<Insert gel pictures here>

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

<Gel picture of Taz 1-pGEM digest (E+B)>

  • 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


Photograph: Gel: Taz1 BB (p-GEM):


<PHOTO TO GO HERE>


  • 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.

Photograph: Gel: BB Vector:


<PHOTO TO GO HERE>



  • 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

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 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 - 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 o OmpC-RFP (Tetr) o 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

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


Sept 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.

Sept 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

Retrieved from "http://2009.igem.org/Team:Brown/Notebook_weekly_Logs/Weekly_Team2_Notebook"