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From 2011.igem.org

September 11th - September 17th

Sunday, September 11

~God bless America -- never forget~

Monday, September 12

Afternoon lab work done by: Nancy Li, Charlie Chung

• Prepared ten 3mL LB cultures (with 3µL carbenicillin) of colonies picked from plates dated Sept 3

- RFP + Avi-Tagged pZE12 in DH5α

- vioA + Avi-Tagged pZE12 in DH5α

- vioB + Avi-Tagged pZE12 in DH5α

- vioE + Avi-Tagged pZE12 in DH5α

- RFP + Avi-Tagged pZE12 in MC4100

- vioA + Avi-Tagged pZE12 in MC4100

- vioB + Avi-Tagged pZE12 in MC4100

- vioE + Avi-Tagged pZE12 in MC4100

- Control tube is just a colony with pZE12 (no insert gene)

- LB with carbenicillin (no inoculation with bacteria)

• Incubating at 37°C in the shaker at Weill Hall
• Cultures are to be lysed for release of natively biotinylated RFP and vio enzymes in characterizing streptavidin-coated microfluidics chips
• Did not extensively search for L-tryptophan (starting substrate in the synthesis of prodeoxyviolacein) in the reagents shelf of our Weill Lab space, but does not appear to be in plain view (may be tucked away in an obscure corner?)

Tuesday, September 13

Afternoon lab work done by: Charlie Chung

Objective: Transfer yesterday's 3mL starter culture into today's larger scale culture (20mL LB + 20µL ampicillin)

• Autoclaved two 1L LB at 1:30pm (~1.5 hour cycle)
• Claire's batch of five autoclaved 250mL flasks are ready for use
• Prepared stock solution of 1000x ampicillin (100mg/mL)

- Weigh out 1g ampicillin sodium salt (Sigma)

- Dissolve in 10mL ddH2O

- Syringe filter (0.22µm membrane) the ampicillin solution

• Stored in the bottom shelf of Weill Lab refrigerator (it's in a 15mL conical -- will later aliquot into Eppendorfs)

Evening lab work done by: Claire Paduano

• Prepared cultures of Avi-Tagged vioA, B, E and RFP in 20mL LB with 20uL ampicillin

Wednesday, September 14

Thursday, September 15

Morning lab work done by: Claire Paduano and Jim Mathew

• DpnI Digestion of PCR Reaction Product

- Retried the PCR deletion method on Avi-Tagged VioA, VioB, and VioE

Avi-Tagged VioA

40.7μL ddH2O

5.0μL 10x PfuUltra buffer

1.0μL dNTPs

1.0μL diluted forward primer (125ng)

1.0μL diluted reverse primer (125ng)

0.3μL dsDNA (20ng)

1.0μL PfuUltra

50.0μL Total

Avi-Tagged VioB

40.6μL ddH2O

5.0μL 10x PfuUltra buffer

1.0μL dNTPs

1.0μL diluted forward primer (125ng)

1.0μL diluted reverse primer (125ng)

0.4μL dsDNA (20ng)

1.0μL PfuUltra

50.0μL Total

Avi-Tagged VioA

40.8μL ddH2O

5.0μL 10x PfuUltra buffer

1.0μL dNTPs

1.0μL diluted forward primer (125ng)

1.0μL diluted reverse primer (125ng)

0.2μL dsDNA (20ng)

1.0μL PfuUltra

50.0μL Total

Run PCR protocol as listed under 'PCR Deletion' in protocols sections

• Transformation via Electroporation

• Add 1.0uL dPN1 to each PCR reaction
• Incubate in thermocycler at 37degC for 1 hour
• After incubation, transform PCR product into electrocompetent cells

- (VioA+Avi-Tag) into DH5α

- (VioB+Avi-Tag) into DH5α

- (VioE+Avi-Tag) into DH5α

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Afternoon lab work done by: Youjin Cho, Charlie Chung

• Gel purification of digested light-lysis gene insert
• Ligation of light-lysis gene insert with pSB1C3 iGEM backbone

Afternoon microfluidics work done by: Maneesh Gupta

• Tested effect of fluorescein with lysate on streptavidin-biotin binding to see if lysate interferes with their affinity

- Used 5:1 ratio of lysate to fluorescein

- Chips used in the experiment were coated yesterday (streptavidin is 1 day old)

- Flow rate = 1µL/min for 50 minutes

Friday, September 16

Saturday, September 17