Hi there, I will appreciate if someone gives me an advice how to clone big inserts in range of 9000 bp...I tried with Gateway system- does not work!
best wishes
Mariya
I'm assuming that your problem is with the ligation and not the transformation- if it is with transformation try using XL-10 cells. Those work nicely for very large constructs. If you can normally clone ~3k bp then I would do subcloning in pieces. You will have to use endogenous restriction sites for the pieces that are not at the beginning or end of your insert.
Hi there, thank you very much for the answers!
Julie, I was considering your suggestion before, but to be honest, I would like to avoid so much of work if possible...without any problem I cloned 2500-3000 bp, but with the 9000 bp I experienced a lot of troubles....I get colonies, but the clones are not correct.
Sebastian, I will read more about your suggestion, Did you succeed cloning big inserts with it?
Take care guys and I appreciate very much your help!
cheers
Mariya
I would also suggest growing your bacteria at 30 degrees or less. We make 4-5000 constructs a year in our group and in our hands growing at lower temperatures solves a lot of problems with unstable or large inserts
Thank you Mark, I could always try!
I try to build up some experience now! 3000-4000 bp is not a problem, but the big once are more stubborn! How ever, I will not give up! Best wishes
Agrose gel purification of the fragment is necessary if PCR is involved. Short unspecific PCR products, even not visible on the gel, will ruin your cloning totally, especially when you try to clone PCR product larger than 5 kb.
Hi there, i always purify the PCR product from the gel....and the reason in the one mentioned above! Thank you very much for the advices !
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