problems in purification of small DNA fragments

carver cathy CCARVER at UGA.CC.UGA.EDU
Wed Nov 30 09:52:39 EST 1994


We have been having some problems in isolating smaller DNA (120bp) fragments fr
om agarose gels. We have tried QIAEX columns, electroelution but with little su
cess. The former did not elute any DNA whereas the latter gave a very poor yiel
d - about 10ng per microlitre. In fact, we have been trying to do site directed
 mutagenesis by PCR as our efforts to do the same using PROMEGA Altered-site ki
t was futile. We could never make ES1301 mutS strain competent to the extent th
ey recommend i.e to over 10 to the power of seven colony forming units in order
 for the kit to work. They suggested us to try electroporation which we have ye
t to try.  Other methods such as RbCl2 and traditional CaCl2 methods did make t
he cells competent but not to the level they suggest.
 
Could someone throw some light on the best possible methods to isolate smaller
DNA fragments from the agarose gels as we need the DNA in sufficient quantities
 to use it as primers in subsequent PCR cycles. Apparently PCR method is workin
g fairly well for mutagenesis as we are getting the expected bands each time bu
t in order to do multiple mutations we have to have enough DNA product isolated
from each cycle to use in the next round of PCR wchich is causing us a great de
al of pain.
 
Thanks for any information in advance which will save us some nightmares.
 
cathy carver
 



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