need help for the transformation
mantei at cell.biol.ethz.ch
Wed Aug 30 03:46:13 EST 2000
<Pine.SGI.4.05.10008291047440.467993-100000 at zathras.chem.bu.edu>, bo
pang <pangbo at chem.bu.edu> wrote:
>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.
If you have random 40mers, presumably many of the plasmid molecules
entering the cells are heterozygotes, in that the two strands of the
randomized region have mismatches. When the plasmid replicates these
segregate and give two different plasmid sequences.
There can also be problems with incomplete deprotection of the oligos.
Bases still carrying protective groups may be seen as something else by
the replication machinery, leading to further mutations.
Department of Cell Biology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland
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