Team:Panama/13 October 2010

October 13

In order to save time and expensive reagents we did a Quick check screening protocol, this experiment will help us find a colony with the transformed bacteria.

Quick Screening Protocol:

100ul of overnight culture 50ul Phenol:Chloroform:Isoamyl alcohol (25:24:1) 10ul Loading Buffer

We mix all the reaction component with a vortex, and then we loaded 20ul into each well of a 1% agarose gel.

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Stock solution preparation of the Primers for Mutagenesis

RhT-g 159 a 1) 5' CGA ATC ACG TGC TAC AGC GCG CCT ACG 3' Resuspend the lyophilized primers for stock soluction (100 uM)

27.9 nM x 10 = 279 ul of water

Working solution (10 uM) 10 ul of stock + 90 ul water

2) RhT-C62T 5' ACC TGC CGC ACT GTA GTT TCT CGC GGG 3'

33.4 nM x 10 = 334 ul of water

Working solution (10 uM) 10 ul of stock + 90 ul of water

3) RhT-g45a 5' CTC GAC CGA ACA CCT ACA GGG CGA CTT CTA C 3'

32.1 nM x 10 = 321 ul of water

Working solution (10 uM) 10 ul of stock + 90 ul of water

We did the miniprep of the cultures, wells 4 and 5

3, 4, 5 and 7 seem to be bigger

We did a PCR following the guidelines on pg. 53 to verify if the cultures of well 4 and 5 actually have the gene Rh1ab so that we can continue with the mutagenesis.

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