Help! PCR primer with CTG repeats
qianfa at mgcheo.med.uottawa.ca
Thu Apr 10 10:58:08 EST 1997
Sorry for the first empty post. I do not know why it went out like that.
Hi, PCR experts:
We have been unsuccessfully amplifying a PCR product and would like to
have some help from the experts on this net.
We are involved in a project to establish stable knock-in human cell
lines (disease gene of interest is replaced by a reporter gene through
homologous recombinations). One of the reporter gene we choose for our
construct is secreted alkaline phosphatase (SEAP2) from Clontech. We
would like to PCR the encoding sequence (about 1.8 kb) of SEAP2 so it can be
subcloned to our construct. Unfortunately there are seven CTG repeats just
following the translation start codon ATG (e.g.
--- ccc acc ATG CTG CTG CTG CTG CTG CTG CTG GGC CTG ---).
The 5' of the primer should have a SalI site, RNA splicing sequence of
the target gene prior to the first ATG. We have made a primer in which
the 3' is ATG (CTG)7 GGCC. We are unable to amplify the right product.
At 50 oC annealing multiple bands are present. At 58 oC, still mutipl
bands. At 62 oC, no product. All of the mutiple bands are smaller than
1.8 kb. The second primer (from the stop codon) used in the reaction worked
very well with other primers.
You can e-mail me directly or post to the net.
Thank you in advance. Your time is appreciated.
| Qianfa Chen Ph.D. qianfa at mgcheo.med.uottawa.ca
| Molecular Genetics Laboratory Tel: 613-738-3926
| Children's Hospital of Eastern Ontario
| 401 Smyth Road Fax: 613-738-4833
| Ottawa, Ontario
| Canada K1H 8L1
More information about the Methods