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Team:Caltech/Notebook/Coliroid
From 2012.igem.org
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Coliroid NotebookTransformed M30109 and R0082, M30109 had 3 colonies and R0082 had none. Back to Coliroid Notebook Calendar 6/25/12 Re-transformed R0082, but with no colonies. Back to Coliroid Notebook Calendar 6/26/12 Ran PCR of R0082 with OneTaq polymerase, gel had no band. Back to Coliroid Notebook Calendar 6/27/12 PCR R0082 with vf2 and vr primers and Phusion polymerase. Gel had confirmed band length. PCR purified and had 34.6 ng/ul. Back to Coliroid Notebook Calendar 6/28/12 Both R0082 and positive control plates had colonies. Transformed RFP (E1010) and lacZ and plated. Back to Coliroid Notebook Calendar 6/29/12 PCR lacZ and pVH001 (mCherry-AAV Victoria's construct) and ran gel with correct band length for lacZ. There is lacZ DNA, but past lacZ transformations have not worked. Miniprepped overnight cultures of R0082 (43.6 ng/ul) and M30109 (28.9 ng/ul). Back to Coliroid Notebook Calendar 7/2/12 Transformed lacZ again and sent R0082 and M30109 for sequencing. Started overnights of R0082, E1010, and MG1655. Back to Coliroid Notebook Calendar 7/3/12 We transformed pSBC13 into Mg1655 on 3 different plates. We put 1 microliter of Mg1655 on 1 plate, 10 microliters on the second plate, and 100 microliters on the third plate, in order to figure out which one would make the best looking plate of red E. coli. We also miniprepped samples of R0028 and E1010. Back to Coliroid Notebook Calendar 7/5/12 We double digested R0082, lacZ, and E1010 (RFP). We ran the digested samples on a gel along with undigested samples of each to make sure the digests worked. The lacZ and the E1010 digests were successful, but the gel results on the R0082 were inconclusive. We also transformed pSBC13 into Mg1655, but the LB was contaminated so we did not plate the culture. Back to Coliroid Notebook Calendar 7/6/12
We started another culture for M1655 since our previous culture was made with contaminated LB. Then we re-digested the R0082 and ran a gel comparing the digested R0082 with the undigested. The digest was successful, so tested the concentrations of DNA in the R0082, the E1010, and the lacZ samples with the Nanodrop. There was not a high enough concentration of the E1010 DNA, so we could not use it in ligation. We ligated the R0082 with the E1010 and plated the plasmid. Also, we ordered primers for mCherry-AAV, mCherry-LVA, and mCherry.
We found that there was growth on the ligation plate and no growth on the negative control plate, which was good. We checked to make sure that the ligation of R0082 and E1010 worked by running a PCR of 6 different colonies on the ligation plate as well as a sample of the undigested R0082 and then running a gel of these things. Back to Coliroid Notebook Calendar 7/2/12 Transformed M30109 and R0082, M30109 had 3 colonies and R0082 had none. Back to Coliroid Notebook Calendar 7/10/12 Started 5 cultures of ligated colony of RFP and R0082 to shake for 5-6 hours and then streak out on plates. Ran gel of lacZ PCR product. Back to Coliroid Notebook Calendar |
