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Splicing DNA fragments

a stretch missing!

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2 replies to this topic

#1 rmbio

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Posted 09 March 2016 - 04:06 AM

Hi all

 

I sliced three DNA fragments by overlap extension PCR for generating a construct for knockout. In the sequenced product, everything is fine except that a stretch of about 250 bp is missing as depicted below.

 

o27mjZQ.png

 

Has anyone ever encountered this? What could be the reason? I visually analyzed the deleted sequence and region flanking it and did not find any repetitive sequence which can cause slippage. Phusion polymerase was used for all the PCRs

 

Thanks



#2 Andrea Fortina

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Posted 20 June 2016 - 12:54 AM

I never encounter this situation before, did you check your template of the 5' homology region before?


Andrea Fortina

Genius only means hard-working for all one's life.

CreativeBiomart, Recombinant protein expert

 

 


#3 827753

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Posted 27 July 2016 - 09:06 PM

http://www.protocol-...osts/40245.html






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