3 replies to this topic

[MikeP]

Hi everyone

I've recently been trying to ligate a 5000 base pair and 10, 000 base pair insert into a pCR-4-TOPO vector (Invitrogen), but am having very little sucess with the ligation step. I've concurrently ligated a 200 base pair fragment to check out my technique and this works just fine. I was wondering whether anyone has attempted to clone fragment of similar length into this vector and sucessfully done so.

Thanks

[pDNA]

There are special ligases available for cloning large fragments, e.g. from Takara (www.takara-bio.us/files/fliyers/d473bbedc8f4d7defd12568a54fd7961.pdf). Normally, cloning of such a fragment should be no problem ...try different ratios of vector/insert as well as different ligation temperatures (RT, 16°C, 4°C) ...and check the ligation on a gel if you get any ligation products ...maybe your restriction enzymes are not cutting well (often the main problem!).

Regards,
                 p

[Vini]

pDNA, on Nov 28 2009, 02:48 PM, said:

There are special ligases available for cloning large fragments, e.g. from Takara (www.takara-bio.us/files/fliyers/d473bbedc8f4d7defd12568a54fd7961.pdf). Normally, cloning of such a fragment should be no problem ...try different ratios of vector/insert as well as different ligation temperatures (RT, 16°C, 4°C) ...and check the ligation on a gel if you get any ligation products ...maybe your restriction enzymes are not cutting well (often the main problem!).

Regards,
                 p

yeah right, more often than not, its the inadequate digestion by RE's that leads to such problems.....i hv never had to clone these big inserts, but i hv seen some of my labmates doing it with normal ligases....So, make sure that REs have really worked well or not....
best.

[Warren]

DRN, on Nov 28 2009, 08:41 AM, said:

pDNA, on Nov 28 2009, 02:48 PM, said:

There are special ligases available for cloning large fragments, e.g. from Takara (www.takara-bio.us/files/fliyers/d473bbedc8f4d7defd12568a54fd7961.pdf). Normally, cloning of such a fragment should be no problem ...try different ratios of vector/insert as well as different ligation temperatures (RT, 16°C, 4°C) ...and check the ligation on a gel if you get any ligation products ...maybe your restriction enzymes are not cutting well (often the main problem!).

Regards,
                 p

yeah right, more often than not, its the inadequate digestion by RE's that leads to such problems.....i hv never had to clone these big inserts, but i hv seen some of my labmates doing it with normal ligases....So, make sure that REs have really worked well or not....
best.

This is a TOPO vector, so it doesn't use the traditional ligase, the ligase activity comes from the topoisomerase, and it is used to clone PCR products (not restriction fragments).  I have not tried to use TOPO vectors recently to clone such large fragments, but I think it should work, if I remember right we had some clones somewhere in between what you are trying to make.  One thing I have noticed is the efficiency seems to drop off alot as the product gets bigger, so your results with a 200bp fragment are probably not applicable.  Then again, since those vectors are so expensive, I don't set up different insert ratios, since I can always get what I want, and the difference between getting 20 colonies and 500 doesn't matter!  Anyway, we would need more details as to what you are doing in terms of PCR, purification, etc etc.  My initial suggestion would be do a lot longer ligation time (rather than 5 minutes) maybe 1/2 to 1 hour, and do everything recommended for "maximum efficiency" in the manual, in terms of transformation and recovery.  Any other recommendations would depend on what your current protocol is....warren..