Meeting+with+Kathy+2-13-12

actually this meeting took place on 2-12-13

docs presented to kathy at this meeting can be found here "C:\kurt\storage\CIM Research Folder\DR\2013\2-12-13\docs for meeting with kathy"

She wants me to check if random oligos are much more expensive than normal oligos. She also said that in the past the 3' nucleotide cannot be random so I should check on this. see questions about ordering random nucleotide oligos

She says I should know how the poly A mRNA was isolated off the top of my head (beads?, column?) She would like me to know the composition of the column for the size fractionation off the top of my head

She wanted me to clarify with Andrey if dynazyme is equivalent to taq (a low specificity polymerase? it is

she would like me to go on the lower end for Tms for pcrs rather than the higher end

determine yield of dna on gels she wants me to keep track of quantitative information on gels (how much ladder is loaded, concentration and amount of dna loaded, etc.)

in general she wants me to get nicer gel images (no shadows, run the gel long enough, etc.). She thinks the gels are very important. She mentioned a new method for avoiding the shadows that I was unfamiliar with. Run a gel with no etbr and no dna in buffer with etbr in it for about an hour before running it with dna. This way the gel and the buffer will have the same concentration of etbr.

She told me a method for finding out the sequence of unknown sequence transcripts. RACE (rapid amplification of cDNA ends) uses TdT enzyme to add dGTP or dCTP or dATP or dTTP to the 5' end of 1st strand cDNA. Then I can use a poly dC or. . . primer to amplify this transcript for the 2nd strand synthesis

After seeing the results of the last cDNA construction (lots of short poly A sequences) we agreed that I could use a random 15 bp hexamers. She thinks I should use them without the in-fusion part first, and then use ones with the in-fusion site later. This idea originally came from a paper Mara found Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA