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#1 2014-07-09 15:42:42

erin.green
Member
Registered: 2014-07-01
Posts: 4

Cloning not working! Any advice?

I just learned of rf-cloning from a colleague last week and decided to give it a try (thanks to your very useful website!). Unfortunately, though I followed your protocol and used the recommended insert and vector concentrations in the second PCR (using the 2-step cycling protocol), things don't seem to be working. I recovered a bunch of colonies following my transformation, but upon screening about 50 by PCR for each construct I'm trying to make (currently trying to make two different plasmids), none contained any insert. Any ideas about what might be going wrong?

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#2 2014-07-09 16:15:31

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 126

Re: Cloning not working! Any advice?

Hi Erin,
Sorry to here it isn't going so well on your first try. I took a peek at some of the projects you've saved to your profile, and one easy thing I can suggest right off, is to include the antibiotics you are attempting to add resistance to in your screening plates. This should prevent any negative colonies, and save you from needing to colony PCR check so many. You can also bump up to Ultra-comp cells in this case, without worry that negative clones are going to swamp your plate.
In terms of the projects themselves, did your megaprimers look good after the primary PCR? Good yield on cleanup? There is some moderately gross complementarity between the plasmid binding side of the two primers in at least one of your projects, so this could be causing some issue. Also, the 3Kb insert is getting somewhat large, which will reduce efficiency in at least that project. Post the exact PCR cycling conditions for me, and the content of the PCR master mix, and I'll see if anything hits me from there.
Take care,
-Steve

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#3 2014-07-09 17:15:39

erin.green
Member
Registered: 2014-07-01
Posts: 4

Re: Cloning not working! Any advice?

Hi Steve,

I haven't tried plating on the double drugs, but could definitely try that fix. The 3kb construct unfortunately doesn't have a drug marker, so I'm still going to have to try to optimize that one. I did only get modest yields from the initial PCR/cleanup for both megaprimers I've been working on. Both products were around 50ng/uL after PCR purification, so I had to add a good bit of megaprimer to the secondary PCR reactions in order to achieve the concentrations that the site recommended.

The mastermix I used for the secondary PCR contained:
4uL 5x Phusion buffer
0.4uL dNTP mix
0.2uL Phusion
0.5uL vector (diluted to ~150ng/uL)
either 5uL megaprimer (for the 1kb insert, which was at about 50ng/uL) or 15uL megaprimer (for the 3kb insert, which was also about 50ng/uL)
and water to make the final volume 20uL

I cycled the secondary PCR using the 2-step protocol, as follows

1. 98dC for 30s (1x)
2. 98dC for 8s
3. 72dC for 3min
4. Go to step 2 and repeat 15x
5. 72dC for 5min

Let me know what you think!
-Erin

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#4 2014-07-09 18:15:57

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 126

Re: Cloning not working! Any advice?

You didn't use TE buffer to elute your megaprimer by any chance, did you? The EDTA would mess things up, although I think most PCR cleanup kits have you elute in water or EB. A good positive control would be to set up some random PCR reaction you know works well, and add in the same volume of megaprimer you used in the 2ndary PCR to see if it bungs up the reaction (assuming you have enough megaprimer to mess around with it like that). Obviously you'll also need the test product to be different size than the megaprimer.
Your conditions look fine to me. You might want to try the 3-step protocol, using an annealing temp of 63-65°C for 30-sec. You could also try some PCR additives (http://bitesizebio.com/2592/better-than … ally-work/).
One other thing you can try, especially for the 3KB project, is to cut the amount of DNA going into the PCR by 5X (i.e.,about 150ng insert, 13ng plasmid).
Fortunately, you have some 1-cutter restriction sites where you want your inserts, so you can always switch to In-Fusion if the RF-Cloning proves too troublesome.
Good luck!
-Steve

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#5 2014-07-12 23:00:49

erin.green
Member
Registered: 2014-07-01
Posts: 4

Re: Cloning not working! Any advice?

Hi Steve,

Just wanted to give you an update. I tried the reactions again with two modifications: 1. After reexamining gels that I ran following the PCR clean-up step, it looked like low levels primers or primer-dimer molecules were still present even after column purification, which may have messed with the secondary PCR. So I re-amplified my inserts and gel purified them prior to the secondary PCR step. 2. I also tried using a new stock of Dpn-i (was worried that our working stock had potentially gone bad). Not sure which modification did the trick, but got TONS of positives the second time around. Thanks for your help!

-Erin

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#6 2014-07-13 15:30:18

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 126

Re: Cloning not working! Any advice?

Wonderful! Congrats.
-Steve

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