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#1 Re: Novel protocols » how to utilize rf cloning protocol for deleting DNA » 2022-02-16 01:26:12

Hey there,
Yes, a deletion is possible. Specify one additional base in the deleted sequence (either with exclamation points in the sequence or by specify the positions in the text fields), then include that single base in the insertion field. This will give you the deletion you desire.
-Steve

#2 Re: rf-cloning troubles » Small insertion into big plasmid, can't get any colony » 2022-01-21 00:32:03

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.

#3 Re: Novel protocols » Get colonies every time » 2022-01-18 09:00:25

Hi Sekissme,
This is great, thank you for sharing. It looks like three significant deviations from the original protocol that should be highlighted (please correct me if there are more):
1) Lower ratio of insert:plasmid (i.e., 3:1 instead of 20:1)
2) Long 2 min annealing time
3) 'Final touch' cycle
Do you feel like you have sufficient data to create a figure for each of these improvements? I've been out of the lab for many years now, so haven't been able to make any technical advances relating to RF-Cloning. If what you claim is well supported, it could be of real value to the community. And possibly publication worthy? Or has there been a new publication(s) that already details all of these?
Again, thank you so much for sharing. This is really exciting.
-Steve

#4 Re: rf-cloning troubles » Small insertion into big plasmid, can't get any colony » 2022-01-18 01:48:26

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.

#5 Re: rf-cloning troubles » RF cloning first PCR » 2021-06-19 10:27:54

Hi Nassiima,
For the 1° reactions, you should be using an annealing temp of ~54°C. While the whole primer may register as having an annealing temperature of 80+°C, remember that the mega-primer is actually a combination of two primers. From the project page, only the green portion of the primer will bind to your insert.
I may be able to assist further if you type out your entire PCR protocol, including all reagents and the exact cycling conditions.
Best,
-Steve

#6 Re: rf-cloning troubles » Unspecific binding of primers to target » 2021-03-06 16:23:22

You should be able to get away with up to 70°C.  Generally, you want to keep the forward and reverse primers within a few degrees of one another, so I'd also update the reverse primer as appropriate.
Good luck!
-Steve

#7 Re: rf-cloning troubles » Secondary PCR transformation not working » 2021-02-14 16:51:47

I think you just need to try it a couple more time, to be honest. I'm not seeing any gross dimer issues and if you're following along with recommended cycling conditions, there's not that much more that will improve things. You can attempt to add a little DMSO to the reaction but you aren't fighting a high GC primer, so I don't actually think it will help. If you can get some higher competency cells, that may be the best bet. Something in the 10^9 range if you aren't there already.
Otherwise, i think the best I can do for you is cross my fingers!
Good luck,
-Steve

#8 Re: rf-cloning troubles » Secondary PCR transformation not working » 2021-02-14 11:36:27

Hello,
Without looking too closely, I see that your target plasmid is fairly large at ~11Kb. Efficiency starts to noticeably decrease above 10Kb. No colonies at least means you aren't fighting false positives right away, so that's a good thing smile
Working in your favour is that you aren't trying to delete a big chunk of the parental and your insert isn't overly large. Before messing around with different PCR conditions, my first suggestion is to make sure your transformation efficiency is high. Are you running controls there? Are you plating out the entire transformation reaction?
If you're convinced the transformation protocol is perfect, then type out the exact PCR protocols you have tried and we'll see if anything pops out.
-Steve

#9 Re: rf-cloning troubles » No positive colony after 2nd PCR,is high T-A concentration a problems? » 2021-01-16 10:14:44

Hi Linh,
You have already mentioned the two main suggestions I would make 1) Increase your annealing temp. This will reduce complications with off target annealing. I would look at something between 55°C and 59°C. 2) Decrease your extension time to 2-3 minutes.
Also, you can reduce the final extension step and cut the cycle number down by 2 or 3.
Good luck!
-Steve

#10 Re: rf-cloning troubles » Second PCR not working » 2020-11-18 01:29:46

Hi Manu,
Ya, I'm not sure what more I can recommend. Have you tried smaller inserts previously and had success? This one is tricky, so if it's your first attempt... Again, I highly recommend In-Fusion. The primers you have are still good and it's way higher efficiency than the RF-cloning secondary PCR.
Sorry for not being much help sad
-Steve

#11 Re: rf-cloning troubles » Second PCR not working » 2020-11-09 23:06:03

Hi Manu,
Your inserts are on the large side. Not to say this isn't doable, but the efficiency is going to be low. My suggestion would be to use InFusion, if possible, otherwise to just make sure you're using highly competent cells, using an appropriate amount of DNA in the transformation, gently centrifuging the cells down and plating everything. You might even want to do 3 or 4 transformations.
For the cycling conditions, you should never go over 15 cycles on the secondary. It causes artefacts to accumulate and further reduces the efficiency. The final extension  can be reduced as well.
Stick with the 65°C anneal and a 5 minute extension. I am not familiar with the KAPA polymerase personally but a brief bit of Googling suggests to me it's a good choice.
I'll keep my fingers crossed for you,
-Steve

#12 Re: rf-cloning troubles » Ordered primers that make the entire insert - overlap on both ends » 2020-08-30 09:08:34

Hi jk,
Yes, you should be mixing these two oligos together in equal molar ratio and move straight into the secondary PCR step. I believe I've found your project in the database but it looks like your megaprimer is actually 114 bp? Regardless, if the plasmid I'm seeing is correct (final size 7383 bps), you're looking at setting up a reaction with 27 ng total of your mixed primers and 88 ng of plasmid. Annealing temp 72C, extension time of 2:26 min, 15 cycles. What's the doi on the paper you've pulled these primers from? I'll have a look at their methods.
Give it another try and let me know how it turns out!
-Steve

#13 Re: rf-cloning troubles » Using electrocompetent cells » 2020-07-14 09:11:06

You should probably treat the input DNA the same as you would following traditional ligation; if you need high efficiency, the salts in the sample will interfere with electroporation. I haven't tried electroporation in this context personally but don't see any obvious reason not to.
My only concern is that many projects see a high number of negative clones, so you may not get a great library. Seems like a 'try it and see' sort of situation.
Best of luck,
-Steve

#14 Re: rf-cloning troubles » Cant get any colonies » 2020-03-03 02:06:04

Hi Asli,
I found three projects attached to your userid, swapping out luciferase for D4, B6, and E3_5.
Your cycling conditions look fine to me, but you are going to have a low efficiency reaction because the plasmid is getting a bit big and the replacement of large fragments usually makes things worse.
Another concern I have is the IRES in between the luciferase and GFP genes. The strong secondary structure can get in the way of proper annealing and extension.
I often encourage people to use In-Fusion, when possible, and I would suggest the same to you. You have a ScaI and NotI pair that will knock out most of the luciferase gene and linearize the parental for the InFusion reaction.
Aside from that, all I can really suggest is poking around more in the forum to see some of the troubleshooting tips people have had through the years. You might have success with some additives.
I wish you the best of luck,
-Steve

#15 Re: rf-cloning troubles » Issues with the Secondary PCR reaction » 2019-08-27 00:16:59

Hi Amit,
I think you may have miscalculated how much of the vector needs to be replicated in the reaction, unless you are trying to delete 5 kb? If you can give me a little more information about your sequence, I can retrieve the project from the database and have a look.
Otherwise, your protocol looks pretty good to me, and the small insert should make things easier. I have high hopes we'll be able to sort you out smile
-Steve

#16 Re: rf-cloning troubles » trouble with 2nd PCR! Please help » 2019-06-23 12:41:59

Hey Hyla,
I would expect that you have caused a lot of high molecular weight artifact to build up in your reaction with this protocol. Try again, but reduce the extension step down to 140 sec and reduce the number of cycles to 15.
And I didn't mean anything more than what I said about the deletion. If you delete a couple dozen base pairs, it doesn't seem to have much of an affect on overall efficiency, but deleting hundreds or thousands of base pairs does. It's just something to keep in mind.
-Steve

#17 Re: rf-cloning troubles » trouble with 2nd PCR! Please help » 2019-06-20 21:12:04

Hi Hyla,
It's difficult to trouble shoot with so little information. There are many tips and tricks discussed throughout the forum, so please see if there is any help there. If nothing seems similar to your issue, then post back with your full protocol and we can move on from there.
One thing that does jump out at me, however, is that you're trying to remove the better part of a KB of parental sequence. My experience has been that big deletions tend to reduce the efficiency of the reaction. Still possible, but harder.
-Steve

#18 Re: rf-cloning troubles » Colony PCR for small inserts » 2018-08-11 14:32:29

Hi Hank,
I wrote that response too quick, without explaining my rational. The issue with Ranjani's example is that the entire insert would have been included in the hybrid primer. Doing a PCR would probably work wether the insert was present in the plasmid clone or not, and will give the same 'positive' result either way. If you have a long insert, then the hybrid primer can definitely be used for QC because there is more information in the plasmid than is present in the primer. Does that make sense?
-Steve

#19 Re: rf-cloning troubles » Colony PCR for small inserts » 2018-07-24 11:16:02

Hi Ranjani,
I would recommend designing a new primer pair instead of relying on the hybrid primers for QC. You'll need them for final sequencing confirmation anyway.
If you want me to have a look at your project, then either PM me the project ID or send me a little more info about the project so I can go looking for it in the database.
Best,
-Steve

#20 Re: rf-cloning troubles » Fake colony » 2018-05-31 15:21:04

This is a difficult project because of the size. I'm not sure there is much more I can do for you.
Are you gently spinning down the cells after transformation and plating all of them? Do you have ultra competent cells you can use?
Just to cover all your bases, you can also run a positive control (no DpnI) to make sure everything up to the transformation is going correctly.
If this construct is key to your work you can also hire a company to synthesize it. I think you would be looking at USD$1500-$2000 for a 17Kb plasmid (maybe less if you don't need all of the elements in that plasmid).
One other possible solution is to cut out the region you are working on and move it into a smaller plasmid, do the rf-cloning reaction, and then swap it back into the large plasmid. I'm pretty sure I found your project in the database, and it looks like you have restriction site options around your insert.
I hope this helps.
-Steve

#21 Re: rf-cloning troubles » Fake colony » 2018-05-25 13:19:49

I hope you're feeling better.
I think you can move ahead with your project with the primers as they are, but in the future I would recommend keeping concentrated stocks (usually 100μM in buffer, then dilute to working conc. in water).
I have my fingers crossed for you. Good luck!
-Steve

#22 Re: rf-cloning troubles » Fake colony » 2018-05-21 13:50:11

That's great about the positive clones! Congrats.
As for the second issue, I'll need you to clarify something for me. You aren't trying to make two mutations in the same reaction, are you?
Let's pretend you want to make a point mutation at position 100 and another point mutation at 400. To do this, you will first make the mutation at 100, purify the plasmid, confirm with Sanger sequencing, and only then will you start to make the second mutation at 400, using the new plasmid as template.
The cycle conditions you provided look fine to me, but why have you diluted your primers to 0.35 ng/μl (i.e., 5 ng / 14.5 ul)? That seems very low, as I usually have them at 5 ng/μl for a single basepair substitution project like yours. If they are diluted in TE buffer, you may be messing up the pH and Mg++ level a bit.
-Steve

#23 Re: rf-cloning troubles » Fake colony » 2018-05-17 01:53:18

Hi again Abdul,
To generate two different single nucleotide mutations, you will need to create specific primers for each. Once you successfully create one, then you can use the mutated plasmid as template to create the other. And you are correct, the primary PCR reaction is simplified.
As mentioned in the Q&A:

Q: When I created a new project there is a note underneath the Reverse Primer that says "The insert is fully synthesized by the primers..."
What, exactly, does this mean?

A: Congrats! You're project is probably a really easy one and you get to skip the 1° PCR ?
Set up the 2° PCR reaction as you normally would but leave out any template and include 5 ng of each primer.
Now run the following PCR program:

Thermal Cycler Conditions
Denature        98°C    30sec    1X
Denature        98°C    8sec    5X
Anneal            55°C    20sec
Extension        72°        5sec
Hold                4°C       
Add your template to the tubes and proceed normally with a full 2° PCR reaction.

I hope this helps,
-Steve

#24 Re: rf-cloning troubles » Fake colony » 2018-05-15 14:01:06

Hi Abdul,
Your destination plasmid is very large, so I'd anticipate you will struggle to get this to work.
How many colonies did you screen? If you are seeing hundreds of colonies on your plate, I would suggest pooling them into groups of 10 to reduce the number of PCR reactions on empty vector colonies. Hopefully you will see one or two pools with the insert, then you can re-screen just that target pool to narrow it down to a clone with your insert.
Is there any chance you can switch to In-Fusion? This will be easier, if you can.
-Steve

#25 Re: rf-cloning troubles » Fake colony » 2018-05-14 13:38:50

Hi there,
Sorry to hear you are having difficulty. I'd like to help, but you haven't provided very many details.
Please list our exactly what you have done (reagent concentrations, cycle conditions, concentration recovered from gel extraction and purity, transformation protocol, etc.), and preferably include the primer sequence so I can have a look at the project itself as well.
-Steve

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