Protocol Online logo
Top : Forum Archives: : Molecular Biology

7kb clone trouble - topo (Aug/06/2002 )

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?


Im not sure that Ive the complete understanding here, but it is impossible to clone a Eco RI site to a TA-site.
They are not compatible.