i have isolated a 7kb fragment of cdna via rtpcr cut the band with ecor1 and am sure i have what i want. following topo ta cloning kit protocol i then am adding this to dh10b cells on a plate containing xgal. i have many blue colonies very few white but am growing these white colonies up then qia preping the dna . the problem is when i do another restriction enzyme digest with ecor1 my fragments do not add up and are very erratic on a 1% agarose gel. is my resuspending pellet via vortexing destroying the cells and thus the plasmid? or is it salt poisoning? maybe need to run through a g50 column?or do the cells just plain not like this and are transforming it to their liking? anybody else have trouble or experience in cloning large fragments?
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