5' RACE troubleshooting

Yarri via methods%40net.bio.net (by yarriofultramar from gmail.com)
Mon Jan 26 06:28:24 EST 2009


Hej!

I am using a SMART RACE cDNA Amplification Kit from Clontech in order
to get cDNA from roots of Datisca glomerata. I have been able to get
few clones from cDNA I amplified with primers that had high Tm values
- as recommended by the protocol. Unfortunately, now I have to work
with more troublesome sequences, where best primers that can be
designed have a Tm value <64C. I was trying to follow the protocol
without much success. Does anyone has some experience with this kit or
some suggestions how to approach the problem? I am new to RACE so I am
not exactly sure how to troubleshoot RACE conditions.
As an example:
A sequencing result:
CATGGAGGATGAGAAACAATATTACTAAAATAACTTTATGTTATGTGCTGTTTGAAGTTTATTTCTCTATGT
TATAACTGCATGCTGCTATCGATCATTGTGAAACAAATCCTTATCAAATATTAATTATATATCTAGCCTTCT
GACTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAMAAAAACAAA
AAAACAAAACAAACAA

GSP2 primer I have designed: CACAATGATCGATAGCAGCATGCAG
NSP2 primer: GTAATATTGTTTCTCATCCTCCATG
Now I am waiting for additional primer that can be used as a nested
one: GTAATATTGTTTCTCATCCTCCATG
Thanks!

                                                       Marian
Płaszczyca


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