Meeting with Kathy 2-13-12
2015-01-13azim58 - 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