Team:UCLA/Notebook/Protein Cages/27 July 2015
Today the new prefix primer (Cage Prefix #2) came in. We resuspended it, made more agarose gels, and ran a PCR with the first-round product containing the his-tag, the new prefix primer, and the suffix. We ran the PCR product on a gel, but got absolutely no bands at all. Tomorrow we will run another PCR with the new prefix primer and the first round suffix to see verify if the new prefix primer works. If it does, then the only problem is still the second suffix primer.
Tyler Lee --Wtleeiv 19:43, 27 July 2015 (CDT)
Phillip's notes
Introduction: A PCR will be done to amplify the gBlock with the primers that incorporate NdeI and XhoI for pET22b. This is in order to determine if a similar problem persists with the non-specific band and low product yield. In addition, buffers for cell lysis will be prepared if the reagents are available.
Procedures:
PCR amplification with primers for pET22b:
Using the NEB Tm calculator, the annealing temperatures chosen were 64C, 68C, and 72C. The following reaction components were combined in a mastermix, and 24uL were aliquotted to 3 reaction tubes. 5x Q5 buffer, 5uL 2mM dNTP, 7.5uL 20uM primer for pET22b forward, 1.875uL 20uM primer for pET22b reverse, 1.875uL 1ng/uL template, 0.75uL Q5 polymerase, 0.75uL ddH2O, 45.75uL
The thermocycler conditions were the same as on 07/24/15. The PCR product was then ran on a 1% agarose gel for 20 minutes.
Results:
Media:2015-07-27 PCR PCquad amplification pET22b 64, 68, 72.JPG
68C and 72C are the preferred annealing temperatures. It seems as though the desired product is present, but it is smeared. The non-specific band appears lower than 250bp, so it may be different than the non-specific bands previously.
The 68C, ad 72C bands were cut out from the gel, and kept in the -20C.
Some notes for Optimizing PCR. https://www.neb.com/tools-and-resources/usage-guidelines/guidelines-for-pcr-optimization-with-thermophilic-dna-polymerases
Conclusions: If the PCquad for iGEM does not end up working, the option of using pET22b vector is still available. Plans for cell lysis are in process. Triton lysis buffer may be available for a more mild detergent. Deoxycholate is a mild ionic detergent.