need help for the transformation

bo pang pangbo at chem.bu.edu
Tue Aug 29 10:29:14 EST 2000


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 
 






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