Re; need help for the transformation

tfitzwater at gilead.com tfitzwater at gilead.com
Wed Aug 30 13:34:10 EST 2000


>From: bo pang (pangbo at chem.bu.edu)
>Date: Tue 29 Aug 2000 - 16:29:14 BST

>I want to construct a DNA library with different sequence. First step,
>PCR, the template contained random 40mers in the middle and normal
>sequence on each end. After the PCR, I could obtain the PCR product
>library and I ligated them into the plasmid vector. The inserted plasmids
>were transformed into the E.coli cell and then the cell grew on the plate
>to form colony.

>For each single colony, I hope that i could get unique plasmid sequence
>from it, it is like I could pick a book from the library. However, when i
>sequenced the DNA purified from the colonies, I
>found that some of my samples are not clean. For example, the vector and
>the normal sequence actually are pretty good, very readable, however, for
>the 40mers part, it gave some random signals. That means this sample
>contains more than one plasmid sequence.

>My first guess is that there was not only one plasmid transformed into
>each competent cell for reason. Because almost each different plasmid have
>different inserted PCR product, the colony I got would not have the unique
>DNA sequence when there were 2 or more plasmid. Is this guess correct?
>Do you have any other explaination for it? Or do you know how I can try to
>tansform only one copy of the plasmid into one cell? Or maybe you have a
>better idea to construct a DNA library like that with small cost.

>Any suggestion is wellcome, thank you in advance.
>Bo

I assume from your description that you are cloning a SELEX experiment.
The first step would be to reduce the amount of DNA you are adding to the
transformation reaction.
When co-transformants are a problem, pick the original colony and restreak
it onto a daughter plate.  The 12-15 generations required to generate new
colonies will allow segregation to occur and will reduce the number of
co-transformants to a more manageable level (perhaps 1 out of 50 colonies).
The daughter colonies should be inoculated into 3 mL of liquid culture (to
allow more segregation) for plasmid prep and sequencing.

Tim Fitzwater
Principal Research Associate
Gilead Sciences


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