5 replies to this topic

[anthony]

I have digested pBluescript II KS- with SapI and have a hairpin of ~15bp with a complementary SapI overhang (starts with 5' - AGC...).  I was trying to ligate the two together, but have been getting a lot of concatamers I believe of pBS to itself.  How can I ensure this does not happen?  Thanks for the help.

[swanny]

anthony, on Sep 11 2009, 02:35 AM, said:

I have digested pBluescript II KS- with SapI and have a hairpin of ~15bp with a complementary SapI overhang (starts with 5' - AGC...).  I was trying to ligate the two together, but have been getting a lot of concatamers I believe of pBS to itself.  How can I ensure this does not happen?  Thanks for the help.

If you phosphatase-treat the plasmid, you will stop concatamerisation. It sounds like you're trying to make linear DNA with a hairpin at each end. Why?

[anthony]

swanny, on Sep 10 2009, 05:55 PM, said:

anthony, on Sep 11 2009, 02:35 AM, said:

I have digested pBluescript II KS- with SapI and have a hairpin of ~15bp with a complementary SapI overhang (starts with 5' - AGC...).  I was trying to ligate the two together, but have been getting a lot of concatamers I believe of pBS to itself.  How can I ensure this does not happen?  Thanks for the help.

If you phosphatase-treat the plasmid, you will stop concatamerisation. It sounds like you're trying to make linear DNA with a hairpin at each end. Why?

I'm not trying to add a hairpin to each end, just one end.  This way I can ligate another complimentary piece to the other end (not a hairpin).

[swanny]

anthony, on Sep 14 2009, 11:37 PM, said:

swanny, on Sep 10 2009, 05:55 PM, said:

anthony, on Sep 11 2009, 02:35 AM, said:

I have digested pBluescript II KS- with SapI and have a hairpin of ~15bp with a complementary SapI overhang (starts with 5' - AGC...).  I was trying to ligate the two together, but have been getting a lot of concatamers I believe of pBS to itself.  How can I ensure this does not happen?  Thanks for the help.

If you phosphatase-treat the plasmid, you will stop concatamerisation. It sounds like you're trying to make linear DNA with a hairpin at each end. Why?

I'm not trying to add a hairpin to each end, just one end.  This way I can ligate another complimentary piece to the other end (not a hairpin).

How are you planning on controlling which end gets the hairpin? And how do you control the reaction so that only one end is hairpinned, as both ends have the same overhang? You might need to use two different enzymes to control that.

[anthony]

It's a SapI overhang so its not palindromic.  I have an oligo hairpin that is complementary to only one of the overhangs.  But in my reaction I am getting concatamers instead of successful ligations.  Which is strange because the hairpin is such a small fragment that I have that in solution in excess to drive the reaction.

[Warren]

anthony, on Sep 15 2009, 09:26 AM, said:

It's a SapI overhang so its not palindromic.  I have an oligo hairpin that is complementary to only one of the overhangs.  But in my reaction I am getting concatamers instead of successful ligations.  Which is strange because the hairpin is such a small fragment that I have that in solution in excess to drive the reaction.

You don't say how you set up your ligation, but a very easy trick to try is: set up everything EXCEPT the ligase and warm up the mixture (with such a small overhang you wouldn't need to warm it up much) and let it cool slowly when it hits around room temp (or cooler if you like) add your ligase.  The idea being, perhaps before ligation (esp. if you set it up on ice) your BS plasmid is already based paired in a concatamer and when you add your ligase your oligo never had a chance to bind.  Easy enough to try anyway.  Warren..