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Team:Nevada/Notebook/Month3
From 2013.igem.org
July1-6
Monday
Christian Designed PCR Primers for KZ144 endolysin.
Christian made liquid LB-amp cell cultures for GFP and OMPA.
Christian finished making the RM medias.
Jon transformed Top10 competent cells with isolated plasmid containing iGEM registered part K523013.
Jon did a digestion of purified OmpA/ GFP pDNA with EcoRI and PSTI.
Tuesday
Christian did a transformation of YFP+INP DNA taken from the iGEM plates and also did a control transformation.
Christian and Jon did a restriction digest of the GFP+OMPA colonies (3, 4, 7, 8) to make sure the cells have both GFP and OMPA.
Jon noted no growth on previous days transformation
Wednesday
Christian did a PCR on the miniprep KZ144 genes with the correct primers.
Christian, Jon, and Jasmine worked on the thank you cards and envelopes to send to businesses for fundraising and donations.
Jon did OmpA/ GFP Gibson assembly.
Jon transformed Top10 competent cells with isolated plasmid containing iGEM registered part K523013.
Thursday
Christian checked the PCR of KZ144 on a gel and found all samples had KZ144 endolysin. The samples were combined to a volume of 50 µl and a PCR purification was
done. The elution volume was 15 µl. This was also checked on a gel to make sure the purification was successful and ready for TOPO cloning.
Christian and Cody found that the LB-AMP plates had gone bad. They made new LB-AMP plates.
Jon observed transformation from the previous day was successful.
Jon ran a miniprep on Plac-INPNC-YFP (K523013) pDNA.
Jon designed and ordered primers for site directed mutagenisis of K523013.
Friday
Christian TOPO cloned the KZ144 and transformed.
Saturday
Christian and Jon did a colony check on 8 colonies from the transformed KZ144 endolysins. Colonies 5-7 were chosen to grow in LB-amp liquid media.
July7-13
Monday
Christian and Jon cleaned the lab.
Tuesday
Jon preformed site directed mutagenesis of K523013 using primers designed previous week.
Jon ran gel of PCR product and verified success of site directed mutagenesis.
Wednesday
Jon ran PCR purification of mutated part K523013. Concentration verified via nanodrop.
Thursday
Jon preformed Gibson assembly of linear mutated part K523013.
Jon preformed Transformation of above part using Top10 cells
Friday
Jon observed that the transformation from previous day was not a success.
July14-20
Monday
Jon preforms transformation again and runs gel of Gibson assembly product to verify adequate concentration.
Tuesday
Jon ran a PCR of Gibson product to guarantee proper concentration. Gibson assembly preformed again.
Wednesday
Jon preformed a transformation using Top10 cells and GA product from previous day
Thursday
Jon noted that after 24 hours incubation the transformation had worked, but only two small colonies were present. Allowed to incubate further.
Friday
Jon line streaked colonies from previous day and grew them in LB CM overnight.
July21–27
Monday
Christian and Jasmine regrew cells with Lyz103 and KZ144 to be used for western blot analysis.
Jon performed a miniprep on liquid cultures and prepared an EcoRI/ PstI digest.
Tuesday
Christian and Jasmine ran a PAGE gel and western transfer for Erwinia endooysin.
Jon ran a gel of previous days digest and confirmed the successful site directed mutagenesis of part K523013.
Jon transformed BL21 cells with GA product
Wednesday
Christian and Jasmine washed and developed western blot for Lyz103 endolysin.
Thursday
Becca, Christian, Jasmine made many different buffers.
Christian and Jasmine ran PAGE gel and western transfer for KZ144 enddolysin.
Friday
Christian and Jasmine washed and developed KZ144 endolysin western blot, but results were no good and they concluded they needed to make new western buffers.
July 28th- August 4th
Monday
Christian and Jasmine ran the PAGE gel and western transfer for KZ144 endolysin. They also made new western buffer.
Christian made 300 ml of LB Broth only, 300 ml of LB with CM, and 300 ml of LB with AMP+CM
Jon preformed site directed mutagenesis of K523013 to remove promoter to yield INPN-YFP
Tuesday
Christian washed and developed KZ144 endolysin western blot. He also ran the PAGE gel and western transfer for Lyz103 endolysin.
Wednesday
Christian and Jasmine washed and developed Lyz103 endolysin western blot.
Jon cloned INPN-YFP into pBAD vector
Thursday
Christian and Jasmine started KZ144 and Lyz103 bacterial cultures to be protein purified.
Friday
Christian and Jasmine started protein purification but lost the cells. Restarted cultures to be purified again next week.
