1st strand synthesis 2-25-13
2014-08-29azim58 - 1st strand synthesis 2-25-13
I plan to synthesize the 1st strand cDNA from my RNA using the
pentadecamer primers with the PrimeScript system.
I plan to perform the 2nd strand synthesis using the PrimeStar polymerase
system as well as Dynazyme.
I would like to set the annealing temperature on the lower end of what is
possible for a pentadecamer. I will refer to Andrey's preferred method
for calculating Tm in CIM
Spreadsheet of Tms for possible 15mers found here
"C:\kurt\storage\CIM Research Folder\DR\2013\2-25-13\cDNA\Spreadsheet of
Tms for pentadecamer 2-25-13.xlsx"
I will choose a temperature at which 3/4 of the bp are As: so 30.8 C
I will also try a higher temperature. The temperature for all 15 bp as Gs
is 60.9 C. I will try a little bit higher around 65 or 68. I may try
several points along this gradient.
What volume of reaction will I have after the 1st strand synthesis?
I will have 20 uL
I will then just use a portion of this for the pcr reactions.
I'll use 2 uL of sample from the 1st strand synthesis for the 2nd strand
synthesis. 5 annealing temperatures will be tried leaving 10 uL of sample
leftover.
pentadecamers have a Tm ranging from 19.9 to 60.9
forward in-fusion sequence tagged pentadecamers have a Tm ranging from
50.6 to 71.1 C
reverse in-fusion sequence tagged pentadecamers have a Tm ranging from
54.7 to 75.2 C
I'll try the annealing temperature at which about 75% of the base pairs
are As: 30.8 C
The annealing temperature for 15 Gs is 60.9 C. I will go higher than this
to 68 C.
Therefore, I will setup a thermocycle gradient from about 30.8 C to 68 C
and pick 5 temperatures to try. After I do the reaction, I expect to see
a moderately strong smear from 0.5 to 5 kb on the gel.
I will use an instance of the dynazyme protocol found here
"C:\kurt\storage\CIM Research Folder\DR\2013\2-26-13\cDNA synthesis\ds
cDNA dynazyme 2-26-13.xls"
see also Dynazyme Protocol
Extention Time Issue
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Need to determine the extension time to use for my cDNA library. I can
refer to the In-Fusion SMARTer Directional cDNA Library Construction Kit
conditions to determine this. In this kit they use the Advantage 2
polymerase. They use an extension time of 6 min with this polymerase.
They use 1 min extension time for a target <1 kb (1min/kb). They use a 3
min extension time for a target 1-5 kb (3min/(5kb)=0.6 min/kb).
If I assume the longer time of 1min/kb, then their 6 min extension time
should allow for amplification of a 6*min*1kb/(1*min)= 6 kb fragment.
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I could also try to look at typical transcript lengths by looking at the
lengths of all of the rna mouse transcripts. See Mouse Transcripts. I
would like to make a regex to reformat this fasta file into columns for
an excel file. See Possible Regex 2-26-13
After looking at the distribution of lengths of RNA molecules (a summary
can be found at Mouse Transcripts), I see that 93.1% of all mouse RNA
molecules have a length less than 6 kb, therefore I will perform the
reactions with an extension time suitable for 6 kb. 6 kb was also the
length that the Advantage 2 polymerase would have covered in the
In-Fusion SMARTer Directional cDNA Library Construction Kit.
===========================================================================
Item: 1st strand cDNA 2-25 KW
Item: D4.2 and D5.2 2-27-13
===========================================================================
After 5 additional cycles for samples without bands, another gel image
was taken.
See annotated gel images here
"F:\kurt\storage\CIM Research Folder\DR\2013\2-28-13\ds cDNA\ds cDNA
2-28-13.svg"
After a discussion with Kathy we decided to take the two lowest annealing
temperature dynazyme samples, combine them, and then electroelute.