I want to clone a 3kb fragment. I use Phusion first to increase the fragment, run a gel, cut the size I want, purify the gel, use Taq to add 3' poly A tail, purify the PCR product, and then do the ligation (TOPO cloning kit) and transformation.
I had very few colonies (sometimes none). I think the competent cells, transformation and plates are all good since the control plate has ~30 colonies. I think either adding the tail or ligation is wrong.
I use beads to purify the PCR products. It's said that polyA tail would gradually degrade, so I wonder if washing with beads would harm the tail? TOPO cloning doesn't work on blunt ends.
Or any advise/ tips on ligation for large fragment (3kb) is welcome. Do I need to incubate for a longer time (4C overnight?) Shaking for a longer time (2h at 37C) before plating? Thank you in advance!
Ling
Hi there,
What is the control plate for your transformation? Suitable competence for cloning purpose should be above 10^6cfu/µg of plasmid (the actual control should be run with 0.1ng of circular plasmid only).
You should definitely run a ligation control (self-circularization of single site digested plasmid) to check ligase activity.
3kb is not that big then classical ligation mix and transformation should be OK (not more than 100ng of plasmid and 3:1 to 5:1 insert :plasmid molar ratio in 20µL, overnight in the fridge is perfect). And run controls in parallel : vector alone without ligase (to measure undigested vector) and vector with ligase (to measure vector self-ligation event).
By the way Taq polymerase is not synthesizing poly A tail at 3' end but only adding one single A.
Hi there,
What is the control plate for your transformation? Suitable competence for cloning purpose should be above 10^6cfu/µg of plasmid (the actual control should be run with 0.1ng of circular plasmid only).
You should definitely run a ligation control (self-circularization of single site digested plasmid) to check ligase activity.
3kb is not that big then classical ligation mix and transformation should be OK (not more than 100ng of plasmid and 3:1 to 5:1 insert :plasmid molar ratio in 20µL, overnight in the fridge is perfect). And run controls in parallel : vector alone without ligase (to measure undigested vector) and vector with ligase (to measure vector self-ligation event).
By the way Taq polymerase is not synthesizing poly A tail at 3' end but only adding one single A.
How do you add the A to your fragment of choice? Just adding Taq polymerase & dNTP to the fragment or run one additional PCR cycle with Taq?
We usually run the PCR with a proof-reading polymerase and add Taq for a last cycle. Of course by this way not all fragments carry the additional A but its always enough to provide several clones for sequencing. We purify our fragments with columns and had never the suspicion that during that process the A was deleted.
Hey, according to my knowledge, there are several blunt-end cloning kit, for instance, the one I use (a Chinese brand) is based on TOPO-clone and it only takes 15min for ligation. Invitrogen has similar products. If the traditional protocol is time-consuming and not very successful, you may want to talk to your PI and buy something new. And it's not expensive at all.
I cloned a 2.8kb long gene using gateway cloning and purified the fragment using gene Jet gel extraction and DNA clean up kit. I found this method more suitable and efficient besides gateway has an upper edge because you can switch species whenever you want.
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