Blunt end ligation problem
John R. McQuiston
zje8 at cdc.gov
Wed Dec 16 08:22:05 EST 1998
I have heard that a exonuclease coprecipitates with sma I and some other
enzymes so some sources of sma I may be contaminated with this. I also don't
like CIP because it seems to greatly decrease transformants regardless, for
whatever reason. Can you try using EcoRV or HincII as the blunt cutter? Also
try cutting a Blue/White vector with EcoRI, filling in and ligating. Then
check the Blue to White cfu ratio to see how many filled in. A complete fill
in should cause a frame shift and hence a white colony.
If possible could you add a EcoRI linker into the smaI site then you have
Any way you put it blunt end ligations stink.
In article <757lm3$ck5$1 at nuscc.nus.edu.sg>, scip7290 at leonis.nus.edu.sg says...
> I am currently facing a problem with ligating Klenow treated
>insert into a CIP-SmaI vector. I have cloned two fragments into this
>vector before, both unidirectional sing two different enzymes. Then I
>tried to clone in an EcoRI insert, so I Klenow treat, and CIP my vector.
>No transformants obtained. I did the following trouble shooting :
>1. Change ligase (2 different ligase)
>2. Change competent cells (check competency with the vector itself)
>3. Tried religation of vector.
> When I tried religating the vector, I could only get a handful of
>transformants. I know that this should not be the case, but I do not know
>where else could be the problem. I changed the water, competent cells,
>ligation conditions etc. I feel that if I cannot religate, something must
>be terribly wrong. Am I missing something important here I-( I refered
>to Sambrooks etc Could someone please just send me a tried and tested
>protocol for blunt end ligation. I will be very thankful.
More information about the Methods