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Team:Cornell/Week 16
From 2011.igem.org
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September 18th - September 24th
Sunday, September 18
Monday, September 19
Lab work done by: Jim Mathew, Youjin Cho, Charlie Chung
- Background
- The PCR site-directed mutagenesis done earlier today should ideally enhance protein expression of VioA, VioB, VioE, and RFP by deleting excessive nucleotides between the ribosome binding site and the ATG start codon in the original DNA sequences
- Work Accomplished
- PCR procedure for deletion technique
- Using 1µl of PCR product of VioA, VioB, VioE and RFP, transformed them into DH5α electrocompetent cells via electroporation
- Put the transformed samples in 37°C shaker for an hour
- Plated the transformed products of VioA, VioB, VioE, and RFP on agar plates treated with ampicillin or carbenicillin (two are on amp; other two are on carb)
- Incubating at 37°C in Olin 303
Tuesday, September 20
Afternoon lab work done by: Charlie Chung
- VioA, VioB, and VioE plates = lots of bacterial colonies, high-density growth
- - Picked three colonies from each plate and started up (3mL LB + 3µL carbenicillin) cultures
- VioA-1,2,3
- VioB-1,2,3
- VioE-1,2,3
- - Incubating at 37°C in Olin 304
- RFP plate = ~5 colonies
- - Picked two colonies and started up (3mL LB + 3µL carbenicillin) cultures
- RFP-1,2
- - Incubating at 37°C in Olin 304
Wednesday, September 21
Morning lab work done by: Charlie Chung
- Transferred 500µL from each culture tube to 20mL (LB + carbenicillin) subcultures
- - Shaking at 37°C in Olin 304
- Miniprep the remaining cultures of VioA-1,2,3 ; VioB-1,2,3 ; VioE-1,2,3 ; RFP-1,2
- - Because of time constraint, was not able to quantify the DNA concentration
- - Sitting in the freezer of Olin 301
Afternoon lab work done by: Nancy Li, Youjin Cho
- After 5 hours of subculturing, transferred all the samples into corresponding 50mL tubes and spun them down at 4500rpm for 30 minutes
- Poured off the supernatant and stored the pellets at -20°C for future use
Thursday, September 22
Afternoon lab work done by: Charlie Chung
- Quantified the Miniprep-purified products with NanoDrop
- - VioA-1 = 68ng/µL
- - VioA-2 = 60.2ng/µL
- - VioA-3 = 88.1ng/µL
- - VioB-1 = 117.6ng/µL
- - VioB-2 = 89ng/µL
- - VioB-3 = 133.6ng/µL
- - VioE-1 = 80.5ng/µL
- - VioE-2 = 73.6ng/µL
- - VioE-3 = 77.4ng/µL
- - RFP-1 = 84.4ng/µL
- - RFP-2 = 56.5ng/µL
- Prepared samples for sequencing submission
- Order Number:10257313
- VioA-1
- 15µL VioA-1+pZE12 (1µg)
- 2µL ddH2O
- 1µL pZE12 forward primer
- 18µL Total
- VioA-2
- 17µL VioA-2+pZE12 (1µg)
- 1µL pZE12 forward primer
- 18µL Total
- VioA-3
- 12µL VioA-3+pZE12 (1µg)
- 5µL ddH2O
- 1µL pZE12 forward primer
- 18µL Total
- VioB-1
- 9µL VioB-1+pZE12 (1µg)
- 8µL ddH2O
- 1µL pZE12 forward primer
- 18µL Total
- VioB-2
- 12µL VioB-2+pZE12 (1µg)
- 5µL ddH2O
- 1µL pZE12 forward primer
- 18µL Total
- VioB-3
- 9µL ddH2O
- 8µL VioB-3+pZE12 (1µg)
- 1µL pZE12 forward primer
- 18µL Total
- VioE-1
- 13µL VioE-1+pZE12 (1µg)
- 4µL ddH2O
- 1µL pZE12 forward primer
- 18µL Total
- VioE-2
- 14µL VioE-2+pZE12 (1µg)
- 3µL ddH2O
- 1µL pZE12 forward primer
- 18µL Total
- VioE-3
- 13µL VioE-3+pZE12 (1µg)
- 4µL ddH2O
- 1µL pZE12 forward primer
- 18µL Total
- RFP-1
- 12µL RFP-1+pZE12 (1µg)
- 5µL ddH2O
- 1µL pZE12 forward primer
- 18µL Total
- RFP-2
- 17µL RFP-2+pZE12 (1µg)
- 1µL pZE12 forward primer
- 18µL Total
Friday, September 23
Afternoon lab work done by: Charlie Chung
- Picked two colonies each from Sean's GFP+AviTag+pZE12 and RFP+AviTag+pZE12 plates and started (3mL LB + 3µL ampicillin) cultures
- - Shaking at 37°C in Weill Hall
Saturday, September 24
Afternoon/Evening lab work done by: Jim Mathew, Nancy Li, Charlie Chung, Claire Paduano
- Preparation of iGEM's pSB1C3 Backbone
- PCR
- Digest with EcoRI and PstI
- CIAP treatment to prevent self-ligation
- Preparation of Light-Induced Lysis Kit Insert
- 3.7ng/µL, 260/230 = 0.05
- Ligation of Light-Induced Lysis Kit with iGEM's pSB1C3 Backbone
- Insert Positive
- 13.3µL Light-Induced Lysis Kit insert (digested with EcoRI and PstI)
- 3.7µL pSB1C3 backbone (digested with EcoRI and PstI & CIAP-treated)
- 2µL 10x T4 DNA ligase buffer
- 1µL T4 DNA ligase
- 20µL Total
- Control
- 13.3µL ddH2O
- 3.7µL pSB1C3 backbone (digested with EcoRI and PstI & CIAP-treated)
- 2µL 10x T4 DNA ligase buffer
- 1µL T4 DNA ligase
- 20µL Total
- Incubating overnight in 16°C water bath of Olin 304
- Insert Positive
- Preparation of GFP+AviTag Insert
- PCR of Sean's GFP+AviTag+pZE12 construct
- Note: Thermocycler in our Weill Hall lab does not seem to be cooling down quickly enough in between cycles. Retried PCR reaction in Olin Hall lab. Tube got warped again.
- - Primers are designed to amplify ("lift off") only the GFP+AviTag and simultaneously add the iGEM prefix and suffix
- Ran PCR reaction product on an agarose gel to check for amplification and to purify the DNA
- - Observed bright bands with migration rate matching 700bp of the ladder
- Gel purification and DNA quantification via NanoDrop spectrophotometry
- - [GFP+AviTag] = 82 ng/µL
- - 260/230 ratio was below 2.0-2.2, suggesting contaminants are still present in gel-purified product
- Digest the GFP+AviTag insert to create sticky ends that match iGEM's pSB1C3 backbone
- 30.3µL ddH2O
- 12.2µL GFP+AviTag (1 µg)
- 5µL 10x NEBuffer 4
- 0.5µL 100x BSA
- 1µL EcoRI
- 1µL PstI-HF
- 50µL Total
- PCR Clean Up of the digestion product of GFP+AviTag
- Ligation of GFP+AviTag with iGEM's pSB1C3 Backbone
- Insert Positive
- 9.3µL pSB1C3 backbone (digested with EcoRI and PstI & CIAP-treated)
- 4.2µL GFP+AviTag insert (digested with EcoRI and PstI)
- 3.5µL ddH2O
- 2µL 10x T4 DNA ligase buffer
- 1µL T4 DNA ligase
- 20µL Total
- Control
- The control reaction tube from the light sensor ligation serves as the control for this ligation reaction as well
- Incubating overnight in 16°C water bath of Olin 304
- Insert Positive
