Gel electrophoresis of Raida's PCR products: Mal-E Gene specific primers

Chris's Gradient PCR gel electrophoresis product. The bands were more specific and showed around 400 bp, which is the size of the desired plasmid. A regular PCR and restriction digest will be run to continue to verify.

Himika's colony PCR of I0500-B0034 construction in pSB1AC3. The bands were expected to be around 1400 bp and they turned out to be as expected. Lane 2 and 3 will be restreaked, plasmid prepped and sequenced.

Friday July 23, 2010

Raida

The first thing I did this morning was run a Gel Electrophoresis of my Mal-E Gene Specific Primer Testing PCR products. I ran it on a 1% gel at 100 V for an hour. Please refer to the image to the side. As it can be seen, the PCR results are positive. All the bands are near the 1200 bp band size, which indicates that it is the Mal-E gene. Furthermore, Lane 7 and Lane 13 show no band because this was the DNA that had the deleted sequence. So the fact that there is no band there is an indication of the fact that the MalE primer is functional and only amplified the MalE gene as it was supposed to. Further testing will be done to assure that the band shows the MalE Gene.

Himika

Today I ran a colony PCR of 11 of the colonies from the plates last night which was I0500-B0034 plasmid switched into pSB1AC3. Most of the colony PCRs seem to work, although the bands are faint. Primers used:

• Bbk_CP_F
• Bbk_CP_R

These two primers anneal 100 bp upstream and downstream of the biobrick prefix and suffix. The expected size of the bands was 1400 bp which was exactly what was seen in the gel. I have decided to forward with B1,C1 which are lanes 2 and 3 respectively.

I also restreaked the two colonies on AC plates and left it in the incubator at 20 C. I also made overnight cultures of the same colonies which will be taken out of the shaker on saturday 24 July, 2010.

Dev This week I worked on the t-shirt designs in Inkscape. The designs are almost finalized. I also helped miniprep the construction of K239000+I13507 today.

Emily

Today I diluted the malE-BBK primers as well as the pRFP-M primers for the site-directed mutagenesis of pRFP. I ran a PCR of malE and malE31 in order to try to Biobrick it. I also ran a PCR of my old construct of I0500-B0034 with the new BBK-CP-F and BBK-CP-R primers. If Himika's I0500-B0034 construct does not give positive reuslts after sequencing, then we may try this one out as a back-up. Raida's PCR of malE and malE31 with gene specific primers as well as the primers that add XbaI and SpeI restriction sites looked really good, so I went ahead and PCR purified the PCR product from the reactions set up with the XbaI/ SpeI primers. Raida also diluted the PCR product that had been run with the gene specific primers and we ran this as a template in two reactions of the malE-BBK PCR.