Team:Panama/13 October 2010
From 2010.igem.org
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.
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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