Design SMC1fs primers to determine orientation and sequence 6-14-13

2015-01-13

azim58 - Design SMC1fs primers to determine orientation and sequence
6-14-13

I need to design primers so that I can do a PCR that amplifies from the
vector to the frameshift region of the SMC1fs sequence so that I can
determine the orientation of the sequence in the plasmid as well as
sequence the fragment to determine whether or not it is in frame.

see also
SMC1fs

Shen sent me some sequences of the wild type SMC1fs sequence and the
frameshift SMC1fs sequence for human and mouse.
These sequences are found here
"F:\kurt\storage\CIM Research Folder\DR\2013\6-14-13\SMC1fs\raw sequences"

I aligned these two sequences and saved the blast output page here
"F:\kurt\storage\CIM Research Folder\DR\2013\6-14-13\SMC1fs\wt and smc1fs
sequence aligned 6-14-13\wt and fs smc1a sequence aligned 6-14-13.htm"

The wild type smc1a mRNA can be found here
http://www.ncbi.nlm.nih.gov/nucleotide/258613891?report=genbank&amp;log$=nu
cltop&amp;blast_rank=1&amp;RID=VR3DTB3301R

From my analysis the fs sequence has all of exon1, two base pairs of exon
2, and then the sequence cuts into exon 4 with the first two base pairs
of exon 4 missing.
Here are my annotated ape files

"F:\kurt\storage\CIM Research Folder\DR\2013\6-14-13\SMC1fs\SMC1A
6-14-13.ape"
"F:\kurt\storage\CIM Research Folder\DR\2013\6-14-13\SMC1fs\SMC1Afs
6-14-13.ape"

However, this is not how Shen remembers that it should be so he said that
he would send me an annotated file.

----------
6-16-13

Shen sent me an annotated file. here

"F:\kurt\storage\CIM Research Folder\DR\2013\6-16-13\SMC1fs\Human and
mouse SMC1A FS rna.doc"

email here
https://mail.google.com/mail/u/0/?ui=2&amp;shva=1#sent/13f49833251822d5

He said that the frameshift sequence is simply exon1 spliced together
with exon4.
see Mouse SMC1a sequence information

Now that I know the sequence, I can design primers that will amplify from
the vector to the sequence. Note that the mRNA could insert into the
vector in either orientation.
cDNA library was constructed with pSMART2IF vector

primer designed can be found here
smc_fus primer

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