I have been trying to clone a PCR amplified full length gene from Drosophila melanogaster in the BamHI-KpnI sites of pCaSpeR4, but my strategies fail every time. The amplicon is 8.6 kb in length. Is there an upper limit to the intake capacity of the vector? Any suggestions??
To clone a 8 kb DNA fragment is readily difficult. Directly clone a PCR fragment in such size adds even more difficulty. Restricting PCR product is always not as good as restricting a bacterium produced plasmid. If you clone your fragment into an easier cloning system, such as PCR cloning kit. Then cut out your fragment with your restricts, clone into your destination plasmid. That will make your life easier. Otherwise, you can use other system to clone such a big fragment, such as gibson cloning system. Good luck.
I used InFusion system to clone in up to 12kb PCR products, but into a different vector (pXL-BACII). With InFusion, you don't need to digest the PCR product, but you need to design the PCR primers in a specific way. Maybe this will help? (http://www.clontech.com/US/Products/Cloning_and_Competent_Cells/Cloning_Kits/Older_Cloning_Kits?sitex=10020:22372:US)
Try to get one of the pCaSpeR plasmids which was modified to work with Gateway cloning. Much easier and faster than trying to succeed with your challenging task.
If you really have no choice, I'd suggest the following:
* Clone your PCR product in some smaller vector, yes, it is an additional step but it will save a lot of time and reagents later
* Cut the cloned PCR product and vector as usual, gel purify, etc. but for the ligation use much more than you would usually use and use a lot of competent bacteria, if you are lucky you might get one colony with the right insert.
Good luck!
Here are some thoughts that might help you:
- An 8 kb PCR fragment is long, but if you have a nice clean band it should work. If you do not have a clean product I would gel purify the PCR product. Alternative: split the PCR product into two.
- If you have a low amount of your PCR product and need to gel purify it, I suggest to use in gel ligation later (having used gels made from low melting agarose to purify the PCR product). Gel purifications kits also work if you have plenty of product; otherwise you might risk to loose/further reduce the amount of your product.
- Do you have problems with a background of false positives (1)? Consider your template for the PCR, i.e. is it a plasmid that has the same resistance as your pCaSpeR4 vector? Then one solution is to clone the PCR fragment first into a (smaller) vector with a different resistance before cloning it into pCaSpeR.
- False positives (2): Does your cut pCaSpeR4 re-ligate, i.e. is not efficiently cut by BamH1 and Kpn1? Then cut the vector into 2 pieces using a unique restriction enzyme (RE) that splits the vector into two pieces of different sizes and use them for cloning/ligation (see attached file).
good luck
you may try the SLIC technique for a big fragment (or several small ones at the same time), cloning gets very efficient.