trouble ligating fusion-PCR into vector - (Jul/11/2006 )
Hi,
I have been reading posts looking for help but now I think I need specific advice. I created a 3kb PCR-fusion attaching GFP to my gene. I added RE sites to each end. I used Platinum Taq High-fidelity to amplify the PCR-fusion. But in 8 attempts I have been unable to ligate the 3kb piece into a plasmid vector. I have tried: Invitrogen's TOPO vectors 2.1 and 4.0 (TA assisted), the Zero-Blunt vector - on the off chance of no A overhangs, pGEM T-Easy, and RE digesting the ends to ligate into my expression vector. No colonies! A few colonies no insert! My vector:insert ratios varied from 1:1 up to 1:10. Positive controls (ligation and transformation) worked, negative controls negative. Antibiotic checked, plates made fresh by me.
Both original PCR pieces used in the fusion were amplified from already existing plasmids. I chose PCR-fusion because the RE and sub-clone method would require several mutagenesis steps and the RE sites were not helpful.
To prepare the insert I butanol concentrate several PCR reactions. Then gel purify (Qiagen kit) and finally drop-dialyze to get rid of any remaining impurities.
I have been cloning PCR fragments for close to 4 years and never run into this much trouble.
Please help if you can!
Thanks,
mcast42
I have a couple questions, which are probably unhelpful because it seems that you've thought of everything:
do you take an aliquot of the Qiaquick/dialyzed insert and check concentration and integrity on a gel? and, do you ever look at your ligation product(s) on a gel?
is it possible for to do direct sequencing of your PCR product to be certain it is what it should be? and, has the plasmid you use as template been sequenced as well?
the only things that readily pop to my mind is that you are losing insert somehow...or that there is a problem with the template?
do you take an aliquot of the Qiaquick/dialyzed insert and check concentration and integrity on a gel? and, do you ever look at your ligation product(s) on a gel?
is it possible for to do direct sequencing of your PCR product to be certain it is what it should be? and, has the plasmid you use as template been sequenced as well?
the only things that readily pop to my mind is that you are losing insert somehow...or that there is a problem with the template?
Whenever I used HIFI Taq I performed a blunting reaction as the product insert said there would be a mix of fragments with blunt end or A overhang after PCR and I thought this would affect my vector:insert ratio if a certain % of the insert was not blunt ended. I then cloned into the Zeroblunt vector.
Did you run the fusion PCR out on a gel?
For my purposes, what protocol did you use for fusion PCR and what size of overhang did you have? I am trying fusion PCR at moment with little success and may try RE method soon.
Which enzymes r u using to digest the sites?
I have a couple questions, which are probably unhelpful because it seems that you've thought of everything:
do you take an aliquot of the Qiaquick/dialyzed insert and check concentration and integrity on a gel? and, do you ever look at your ligation product(s) on a gel?
is it possible for to do direct sequencing of your PCR product to be certain it is what it should be? and, has the plasmid you use as template been sequenced as well?
the only things that readily pop to my mind is that you are losing insert somehow...or that there is a problem with the template?
Whenever I used HIFI Taq I performed a blunting reaction as the product insert said there would be a mix of fragments with blunt end or A overhang after PCR and I thought this would affect my vector:insert ratio if a certain % of the insert was not blunt ended. I then cloned into the Zeroblunt vector.
Did you run the fusion PCR out on a gel?
For my purposes, what protocol did you use for fusion PCR and what size of overhang did you have? I am trying fusion PCR at moment with little success and may try RE method soon.
Hi,
For the fusion PCR I used the protocol outlined by Yu 2004 (double-joint PCR). I found the key step is to run a 'no primers' rxn for 10 cycles. Then use an aliquot from that - 4ul for mine to run the fusion-PCR with primers. My overhang was 25nt and I calculated the Tm based only on the actual primer match. Thus my primers were over 50nt with a Tm around 65-69C.
As to my problem, I tried sequencing the PCR-fusion but the sequencing rxn was bad. I ran the fusion-PCR on a gel to be sure I had a clean band and it was the right size.
The blunt vs. A overhang is why I tried both kinds of vectors. I am now trying the RE - ligation again on the off chance it will work this time.
mcast42
do you take an aliquot of the Qiaquick/dialyzed insert and check concentration and integrity on a gel? and, do you ever look at your ligation product(s) on a gel?
is it possible for to do direct sequencing of your PCR product to be certain it is what it should be? and, has the plasmid you use as template been sequenced as well?
the only things that readily pop to my mind is that you are losing insert somehow...or that there is a problem with the template?
Hi,
I started running the extract out on a gel precisely to check 1) did I get anything 2) is it clean and 3) to get a concentration using the imaging software.
I am starting to think the problem is actually with the 5' end of the insert. I was able to clone the GFP fragment separately into a Zero-Blunt Vector but not the gene of interest piece nor the PCR fusion of GFP and gene of interest.
I PCR checked the ligation reactions using M13F&R and no insert.
Sigh...
Thanks for your ideas,
mcast42
I have the GFP-F2A sequence in Zeroblunt and I have a clone which is in the orientation allowing me to do a double digest using the blunt ended EcoRV (from Zeroblunt) and the sticky ended PspOMI (from F2A sequence). I then have amplified my other fragment which is F2A-gene with a proofreading polymerase and will digest this with PspOMI alongside my double digests to confirm I am getting a good PspOMI digest. I have tried this once and after ligation (using NEB Quick ligase) I ended up with a fragment a lot larger than expected so the blunt ends (I think) must be annealing.
Previous to this I had tried fusion PCR using the additional step with the 10 cycles with no primers to aid in annealing of the two fragments (which have a 19bp complementary sequence) but I never was able to achieve a fragment at the end of the second PCR that was the correct size and was always ablt to see the two starting fragments clearly in the gel.