received random hexamer tumor library clones 2-7-13

2014-08-29

azim58 - 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.

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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.