rout@quads.uchicago.edu (Mark Routbort) (09/12/90)
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).
The sequence of steps on the above control was:
1) Cut CsCl-pure DNA (of a clone that has a 3 Kb insertion into SmaI site of
Bluescript) with Asp718I, XbaI, and StuI (which cuts not in vector but in
insert so I could distinguish vector fragment from insert). phenol/CHCl3/EtOH.
2) T4 polymerase to hopefully blunt. phenol/CHCl3/EtOH.
3) Calf intestinal alkaline phosphatase. Heat inactivate in presence of
EDTA (15' at 65 deg C). Prep gel. Cut out and elute 3 Kb Bluescript fragment.
Phenol/CHCl3/EtOH.
4) Rephosphorylate with T4 polynuc. kinase.
5) Use 100 ng of DNA in kinase buffer to ligate with 10 units T4 ligase
overnight at 12 deg C.
6) Transform into XL-1 competent cells. No colonies next day.
I've never had problems ligating or transforming before using cohesive or blunt
ends (directly from enzymes), so I think the problem lies either with the
kinase or the polymerase (both brand new). Has anyone had similar problems and
resolved them? Unfortunately I _can't_ cut the PCR fragment and ligated with
cohesive ends on both sides, although I can do so on _one_ side.
Thanks for any tips, insights, and sympathetic commiserations.
--
Mark Routbort Due to circumstances beyond my control,
rout@midway.uchicago.edu There will be no big parade this Sunday.
-- Colonel Scheisskopf in Catch-22arenas@citiago.bitnet (Jaime Arenas) (09/14/90)
> 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@CITIAGO.BITNET ARENAS@IAGO.CALTECH.EDU (NO, there are no misspellings)