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Hi, and thank you for designig this online tool, it is very useful and well designed.
I'm currently working on this project
https://www.rf-cloning.org/rf_cloning_project.php?proj_id=04c2c62c2186270c945ee0e201ca24d7
The insert is fully synthesized by the primers, so this is the protocolo that i'm following.
Since i'm using the Q5 DNA polymerase, i'm setting up the reaction setup according to NEB website:
Q5 buffer 10uL
10mM dNPTS 1uL
10mM FP 2.5uL
10mM RP 2.5uL
Template DNA /////
Q5 Dna pol. 0.5uL (0.02U/uL)
DMSO 2.5uL (5%)
Water up to 50uL
The first PCR, without the template:
Denature 98°C 30sec
Denature 98°C 8sec
Anneal 55°C 20sec 5X
Extension 72° 5sec
Hold 4°C
Then, I add 10ng of template DNA and start this second PCR reaction:
Denature 98°C 3 min
Denature 98°C 30sec
Anneal 72°C 30sec 25X
Extension 72° 10min, 50sec
Final extension 72°C 120sec
Hold 4°C
After running the PCR product on 1% agarose gel I don't get any band, and if i try to transform, after DPNI digestion and transfrormation into DH5alpha competent cells (and i've tested them, they work), i do not get any colony.
I would imagine that being quite a big vector (13kB) could be a issue...What can I change? Am i getting something wrong?
Thank you!
Last edited by Sybr_Green (2022-01-18 09:27:27)
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Hi Sybr,
The small insert will help, but that is a rather large plasmid.
A few bits of info and advice:
- Don't expect to see much by running a gel on the secondary PCR. No band means nothing.
- You're running the secondary PCR for too many cycles. Cut it back to 15.
- Your extension time in the secondary is long. Google isn't immediately telling me what the processivity of Q5 is, but NEB asserts it's 'fast', so I'd dial it way back to what the RF-Cloning tool is suggesting (4:18 min extension)
- Do as many transformations as you deem appropriate from the volume of secondary PCR you have after the run, and then plate everything (i.e., gentle centrifuge into a soft pellet, resuspend in a reasonable volume, and spread it all on a plate).
- Include a positive control -> take a sample before DpnI treatment and use it to transform. You should see many colonies from that.
These are the easy suggestions I can make based on your project and protocol. I'll cross my fingers they will be enough.
Good luck,
-Steve
Edit: It seems like your timing was most opportune. Please see this new post that includes additional suggested protocol modifications.
Last edited by Steve Bond (2022-01-18 09:09:19)
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Hi Steve, thank you very much for your answer and suggestions.
I've followed them but still can not get any colony on the plate.
I've also tried to transform without DPNI digestion, getting plenty of colonies. This means that the DPNI digestion is working, since i don't get colonies after I digest and plate the PCR product, right?
Hi Sybr,
The small insert will help, but that is a rather large plasmid.
A few bits of info and advice:
- Don't expect to see much by running a gel on the secondary PCR. No band means nothing.
- You're running the secondary PCR for too many cycles. Cut it back to 15.
- Your extension time in the secondary is long. Google isn't immediately telling me what the processivity of Q5 is, but NEB asserts it's 'fast', so I'd dial it way back to what the RF-Cloning tool is suggesting (4:18 min extension)
- Do as many transformations as you deem appropriate from the volume of secondary PCR you have after the run, and then plate everything (i.e., gentle centrifuge into a soft pellet, resuspend in a reasonable volume, and spread it all on a plate).
- Include a positive control -> take a sample before DpnI treatment and use it to transform. You should see many colonies from that.These are the easy suggestions I can make based on your project and protocol. I'll cross my fingers they will be enough.
Good luck,
-SteveEdit: It seems like your timing was most opportune. Please see this new post that includes additional suggested protocol modifications.
Last edited by Sybr_Green (2022-01-20 09:51:45)
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Shoot. The DpnI-free control just gives you an idea as to the upper limit of colonies you could expect. Assuming you're going to lose 99% of the parental plasmid in the process, if you only see a couple hundred colonies from the positive control, then that would be an indication you are going to struggle to get anything from the DpnI treated sample.
Regardless, did you try any of the suggestions in the new post I linked to? There were some interesting ideas there.
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