Dear community,
I am working with a HUGE protien, in a plasmid we received from collaborators. It is 20,880 bp in size, and the gene of interest is aprox. 15,600 bp. Thankfully the received plasmid was Gateway compatable, in a Flag tagged vecor with the appropriate attB1/attB2 sites and made it easy to create a donor vecor and shuttle into a GFP.
But here is my problem: We want to remove a c-terminal fragment of the gene. You can imagine this is difficult because of the size and trying to avoid multiple cut sites. There is an FspAI site that cuts once right before this C domain I want to cut out, and a NotI site directly after the stop codon at the end of the gene.
Using SnapGene simulation- FspAI produces a blunt end, and NotI produces a 5' overhang. When simulated, Not1 site is preserved, but the stop codon (everything still in frame) is slightly downstream just after the attB2 site now (shouldn't be a problem)
Here is put simply what I have done:
1. Thermo Scientific enzymes - both in Buffer O (Cut 100%) overnight- Run 1% gel- Bands look perfect - I cut out top band as that should be my vector, and lower band is the c-term to remove
2. NEB quick blunting kit on the DNA of the gel extracted top band - this should remove or fill in the 5' overhang and leave me with now two blunt ends
3. Tried regular T4 ligase, but have moved on to a more blunt-ended specific ligase by NEB - Blunt/TA Ligase master mix (which has PEG- better for blunt ended ligating)
Followed all directions to a T. Now trying different variations of amounts of DNA, blunting first, then gel purifying, then ligase. Not getting any colonies! Normally people ask how to prevent recirculating of the vecor, and that is ALL I want to do!
Please help if you have any advice, or if you've done this. Thank you so much!
Hi Katie,
It may not help you with your immediate problem however, I generaly opt for a PCR based strategy to remove any gene fragment in a plasmid.
Basically yo design a primer pair that will amplify away from the gene fragment to be deleted. In a circular DNA you would expect that the whole plasmid will be amplified. The new DNA strands come together by the corresponding 30 bp complementary overhang.
The original plasmid DNA is destroyed by DpnI activity and the rest of the DNA should constitute only the PCR products, which can be used for transformation and subsequent screening of the mutant plasmids.
Best
Sudeep
Gibson/USER strategy will do your job. Split 3 to 4 pieces of PCR fragments, do PCR, then Gibson/USER them together.
Dear Sudeep Kumar and Ziguo Zhang thank you for your suggestions!
For doing PCR away from the fragment to be deleted, the remaining vector is still 18,000 bp and I think that is too large for the PCR to amplify properly? Do you have experience with this with such a large plasmid?
Ziguo Zhang I have looked into some the Gibson assembly- though I am confused by the actual steps. I think the max fragments even the Platnium Taq DNA Polymerase can PCR is a little more then 3,000 bp, so I would have to make 6-7 primer pairs (because each would have to include a 15 nucleotide overlap) put each pair in individual pcr tubes with the origional plasmid as the template, run PCR, then gel purify? Or add the PCR products directly together in the Gibson assembly buffer and hope that they all assembly together correctly?
I love the suggestions, I'm just still confused as to why cutting out my fragment and ligating the vector back together is proviing to be so difficult? It seems like it should literally be "cut and paste"
Thank you!
It is not true that the PCR length limitation. Phusion polymerase can easily amplify > than 10kb fragment. 3-10 kb can be routinely done without much difficulty. I routinely made plasmid larger than 20 kbs, with the biggest about 50 kbs so far in my hand with USER and/or Gibson methodology. Traditional cut and ligation, especially blunt ends ligation, is much less efficient for such large plasmid handling.
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