Help! PCR primer with CTG repeats

[Qianfa Chen]

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
|