Cloning problems-supercoil

lvisser at lvisser at
Tue Sep 9 14:35:08 EST 1997

Just another cloning problem. I blunt-end cloned ( into the SfrI site) a 1400 
bp PCR fragment amplified from malaria cDNA into the Stratagene pCR-Script 
vector (pBlue based, 3000 bp). On isolating the recombinant plasmid in a 
miniprep, I could cut out my insert (using PvuII) perfectly.  But upon trying 
to get the plasmid clean enough for ABI sequencing, I had a plasmid running at 
1500 bp!!. At first I thought it was due to recombination in the DH5a cells 
that I used (giving rise to the smaller bands)  but I had the same problem 
once I retransformed into SURE cells. I then started looking at the 
purification method (High Pure plasmid isolation Kit from Boehringer) and 
tried to precipitate my miniprep plasmid with PEG and glassmilk -same results 
of a smaller plasmid. I eventually came to the conclusion that the plasmid 
supercoils upon futher purification/concentration (proved with EtBr 
intercalation and observing the shift in mobility) possibly due to the A/T 
rich malaria genome (80%)?.

My big problem at the moment is to try and sequence the 1400 bp insert. I have 
tried to denature the super-supercoiled recombinant plasmid at 96 degrees for 
5 min. as well as alkaline denaturation as used for manual sequencing. Both 
these gave very weak base signalling with the ABI sequencing, as if too little 
template was present.  

What to do next???? 

Thanx in advance

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