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CAROL
Colony PCR and Enzyme Digestion of Mutated Plasmid
- Attempted to perform colony PCR and enzyme digestion with XbaI on the mutated plasmid with a positive control (non-mutated plasmid)
- Results showed that plasmid does NOT contain the XbaI site (the 0.6% agarose gel showed that there is only one 6KB band from the colonies whereas the positive control showed two bands)
- Will try to isolate higher concentrations for sequencing.
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CHINMOYEE
Research more on Stochasticity
Looked into Papers :
- Imapct of Stochasticity on gene regulatory networks
- Analysis of Noise in AI-2 Circuits
- Stochasticity in Transcriptional Regulation: Origins, Consequences,and Mathematical Representations
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EMILY
Descriptive Title of What You're Doing
- Purpose: To verify that BBK LuxOD47E has been successfully cloned into the psB1AC3 vector.
- Performed a Colony PCR on LuxOD47E in psB1AC3 vector colonies from last week. Only digests done with XbaI / PstI enzymes produced colonies on plates, so these were the only colonies used.
- Used p Taq and LuxO forward and reverse primers. Cycling conditions were as follows: 94 C for 6 minutes, 36x (94 C for 30 sec, 55 C for 45 sec, 72 C for 90 sec), 72 C for 10 minutes and hold at 4 C.
- See gel photo below. Lanes 1-6 are LuxOD47E cut with XbaI / PstI, colonies 1-6, Lane 7 is a negative control with ddH2O.
- Results: Nothing was amplified. This was because I used the wrong buffer. Will proceed by re-running the Colony PCR with the CORRECT buffer (10x PCR Buffer- Cl2).
- Performed colony PCR again with the proper PCR Buffer this time. PCR products were visualized on a 1% agarose gel run at 120 V with Generuler 1.0 KB+ DNA Ladder. Lanes 1-7 are LuxOD47E in psB1AC3 cut with XbaI / PstI. Lane 8 is a negative control with only Mastermix + ddH2O.
- See gel photo below.
- From this gel, it looks like LuxOD47E is in the vector as we see the appropriate band size of approximately 1.4 KB.
- Prepared overnight cultures of the colonies, will isolate plasmid tomorrow.
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JAMIE
Construction verification
Colony PCR could not be used as a means to verify due to the presence of intrinsic terminators (B0015) within the plasmid backbone. If B0015 gene-specific primers are used, multiple bands will likely appear. Instead a NotI digest coupled with a size control will be used as a verification. Standard manufacturer's specifications followed.
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JEREMY
PCR of luxPQ in psB1AK3 using BBK-CP-F/R and gene specific primers
Purpose: to verify presence of LuxPQ in psB1AK3. Colony 4 of luxPQ in psB1AK3 was used as a template and pTaq DNA polymerase was used. LuxPQ-F/R gene specific primers and BBK-CP-F/R primers (that anneal just outside the BBK prefix and suffix) were used. Conditions were as follows: 94ºC for 2 minutes; 36X (94ºC for 30 seconds; 55ºC for 45 seconds for BBK-CP-F/R primers (53ºC for 45 seconds for LuxPQ F/R primers); 72ºC for 4 minutes); 72ºC for 10 minutes; held at 4ºC. The products were then run on a 0.8% agarose gel (see below). Again, we are looking for a band size of just under 4kb, which we do not see here, and we must thus restart from luxPQ in TOPO.
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KEVIN
Restreak
Restreak of R0040, B0015, and J13002 were done to grow more of verified single colonies.
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PRIMA
Understanding Lab and Continuation with marketing
I learned the purpose and procedures for restriction digest from shadowing Jeremy. After hanging out with in the lab for some time, I began understanding what PCR was used for, when PCR needed to be done, what's the water bath for and much more.
Carol also taught me what phosphatase is, when it should be done and even showed me how she was doing it.
I kept following-up with the list of companies that Fahd and I had made in the beginning of the year. Many companies have turned down our proposals due to financial constraints. On the bright side, we decided to target mostly pharmaceutical and biotech companies, many of which are considering our sponsorship proposal.
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