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Jaime Arenas arenas at IAGO.CALTECH.EDU
Fri Sep 14 03:53:56 EST 1990


>     I'm looking for someone with experience cloning medium sized (500-1000 bp)
> PCR fragments into a vector for screening.  I'm having troubles getting
> transformants to screen.
>      I discovered through a control experiment that Bluescript vector that I
> had cut, blunt-ended with T4 polymerase, phosphatased with CI alkaline
> phosphatase, and then kinased with T4 polynucletide kinase would NOT yield
> Amp resistant colonies.  Unfortunately I don't have the cut, blunted,
> unCIAPed vector to determine if the problem lies with blunting or with
> kinasing (although either is a problem, since I have to both blunt and kinase
> the PCR fragements).
 
 
Mark,
 
I had similar problems trying to clone PCR products in bluescript.
I was trying to blunt-end ligate PCR product into the SmaI site of BS. However,
It never worked. Although I never knew for sure what was wrong and to make a
long story short, here's what I found and how I did come out of it:
 
1.- I found that "blunt ending" PCR fragments with T4-pol was not needed indi-
cating that there was enough blunt ended molecules in the PCR product.
 
2.- Always had troubles when cut Bluescript with SmaI. Then changed to EcoRV and
 
worked just fine.
 
3.- After treatment of EcoRV vector with alkaline phosphatase, I removed it
by phenol/chlor (ph 7.0) extraction and three chlor. extractions followed by
ethanol precipitation.
 
4.- After PCR I phenol/chor. and etOH pp.  products as above and phosphorylated
them with kinase.  Then isolated kinased PCR products by running Nu Sieve LMP
agarose gel (SeaKem) and cut band out.
 
5.- Blunt end ligation works just fine adding an aliquote of the melted agarose
slice up to 0.3% agarose in the ligation reaction.  For this, melt agarose,
mix all reagents except enzyme in another tube and heat to 50-55 celcius.
Mix melted agarose and pre-warmed reagents. Put at 37 degrees and add enzyme.
Leave at 37 degrees 10-15 minutes and then sit at RT for 3 hours or more.
 
6.- Although this was enough for me. For higher yields purify PCR products
 away from agarose
 
 
 
P.S. If you are using Blue/white selection, never mind the color of the
colonies when using Bluescript I.
 
Good luck,
 
 
---------------------------------------------------------------------------
 
Jaime Arenas, Ph.D.                       There is no places,
Division of Biology 147-75                there is beginnings and ends,
CALTECH                                   but nothing is ever forgotten.
Pasadena, CA 91125
 
ARENAS at CITIAGO.BITNET
ARENAS at IAGO.CALTECH.EDU                  (NO, there are no misspellings)
 




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