received+random+hexamer+tumor+library+clones+2-7-13

associated plan for these results cDNA Library Random Hexamer Plan 12-12-12

sequencing info found here "S:\Research\Cancer_Eradication\Discovering tumor specific antigens\Tumor cDNA Library\2-7-13 Random Hex"

Sequencher file found here S:\Research\Cancer_Eradication\Discovering tumor specific antigens\Tumor cDNA Library\2-7-13 Random Hex\Random Hex Analysis\original sequences

Summary excel sequence here "S:\Research\Cancer_Eradication\Discovering tumor specific antigens\Tumor cDNA Library\2-7-13 Random Hex\Random Hex Analysis\Random Hex sequence info 2-7-13.xlsx"

Note that "vector" in this data is pSMART2IFD vector

Note that I started aligning these sequences in sequencher. However, I found that I was spending quite a bit of time manually fixing the alignment later. I found that the online Clustal Omega alignments were working much better. Comparison of Sequencher and Clustal Omega

A Summary of the results can be found here "S:\Research\Cancer_Eradication\Discovering tumor specific antigens\Tumor cDNA Library\2-7-13 Random Hex\Random Hex Analysis\Hex analysis summary 2-7-13.docx" Text version of summary for searching 2-7-13

Info from these results -I seem to have designed the poly T to be attached to the wrong in-fusion sequence <- actually the poly T primer I used from the kit is correct. The poly T primers I ordered are also correct. -All of the poly T sequences are very short -The sequencing primers seem to be far too close to the region of interest to get good sequence

The fragments amplified from the random hexamer primers seem to be okay and amplified mouse transcripts.

Why are all of the poly T sequences so short?

When I constructed this cDNA the 3' In-Fusion SMARTer CDS primer (poly dT primer) was used (see 1-24-13 of notebook). The 3' In-Fusion SMARTer PCR primer was used during an additional 5 cycles of the double stranded cDNA synthesis (see 1-29-13 of notebook).

I think I know the reason that the poly A sequences are so short. These primers only have two base pairs for priming to the RNA transcript. Normally this kit uses the SMARTScribe reverse transcriptase with some unusual thermocycling conditions. I did not use the SmartScribe reverse transcriptase. Instead, I used the PrimeScript reverse transcriptase with the thermocycling conditions for that polymerase. I think this can explain the short poly T sequences I obtained.