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

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.

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.