How to Create Tetramers or Hexamers from a 96-Base Long DNA Sequence?

[karthik3k]

I have a DNA sequence of 96 bases long.
I want to make a tetramer/hexamer of this sequence.
Is there any standard protocol available for this?

 

[iansmith]

It is the best thing I could found

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WBK-45PMJV4-2H&_coverDate=11%2F30%2F1994&_alid=226736773&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=6713&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=782f92ac3f85811a2a132b8525f70882

 

[Monique]

I once did it by cloning the sequence in a vector, make lots of it by transforming some bacteria, cut it out with sticky ends, ligate, run on gel and cut out the correct size (you'll see a ladder), put back into a vector, put into bacteria and then you can do a pcr if you wish. It's a LOT of work and you need to watch out that you don't form circular plasmids during the ligation step.

You could look up some LONG SAGE protocols, they have a step where they form concatamers.

 

[karthik3k]

circular? yeah... that's wat i was worried about...

How can i avoid it?

N i couldn't have the paper specified above :( ( No subscription...!)

 

[karthik3k]

actually i have got a sequence and its complementary sequence with extra 4 bases on both the sides(5' n 3'). n i want 2 create tetramer of this sequence thru ligation.

Is it possible ?

The orginal sequence is of less than 80 bases.

Any ideas ?

thanks in advance! :)

 

[Monique]

I can not get to the article myself.. you could try your university library and see if they can get it for you.

I found it all to be very tricky, but you must try to do the ligation at a very high concentration of the short pieces of DNA: it will be more likely that a short piece of DNA will attach itself, than it would be for the concatamer to close itself.

Try looking up articles on long sage and see how they do this step.

I tried hard to get a concatamer of 100 and 200 bp building blocks.. I did get the ladder, but was unable to clone it: because the concats were circularized.

Another option might be to use CIP (calf intestine alkaline phosphatase) on the DNA, it will dephosphorylate the one end of DNA so that it can't circularize.. good luck ;)

 

[Monique]

ps, try partial digestions of the circularized concats (if you've got enough).. that might give good results too.

 

[iansmith]

I can not get to the article myself.. you could try your university library and see if they can get it for you.

I have send the article couple days ago