need help for the transformation
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Tue Aug 29 11:21:45 EST 2000
In article <Pine.SGI.4.05.10008291047440.467993-100000 at zathras.chem.bu.e
du>, bo pang <pangbo at chem.bu.edu> writes
>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.
Secondary structure of the 40mer region blocking the sequencing?
>That means this sample
>contains more than one plasmid sequence.
If the sequence is in any way lethal you could be growing a plasmid
population with different sequences.
>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?
I would think this is unlikely. Usually the incompatibility regions of
the plasmid will stop that occurring.
How are you doing the sequencing i.e. transform library, pick colonies
and streak out on plates. Pick a single colony from each plate, grow up
in liquid, miniprep and sequence. From your first paragraph I am unsure
if you are just transforming and sequencing directly from a resulting
colony. If so you may well have a mixed culture within that supposed
clean colony. Plating again and picking a single colony would at
hopefully give you single isolates.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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