Wrong or No fragment in Directional Cloning Vector - (Mar/24/2016 )

Dear helpful community,

 

I am having trouble PCR-amplifying and cloning a ~2.5kb fragment into the pET101 directional cloning vector by Invitrogen.

 

PCR conditions:

-Q5 high-fidelity polymerase (produces blunt ends)

-Gene-specific forward and reverse primers Tm ~60C

 1. 98C initial melting temp, 2 minutes

 2. 98C melting temp, 40 seconds

 3. 55C annealing temp, 30 seconds

 4. 72C, 3 minutes (loop to step 2 34 times)

 5. 72C, 5 minutes (final extension)

 6. 4C, hold

 

After cycling, I have very strong products that appear to be about 2.5kb in size, with only faint secondary bands that I presume are the original plasmid in different conformations .

 

I then excise my bands form the gel, combine excised fragments, and purify using a basic kit and end up with about 50ng/ul DNA of fairly high purity.

I then ligate the purified fragment into Champion pET101 Topo directional cloning vector according to the protocol by Invitrogen, and finally transform.  The next day, I see about 30-50 colonies on each plate

 

The problem(s): 1) Running colony PCR with T7 forward and reverse primers shows that most clones are missing the 2.5kb insert, and 2) those that appear to have an insert are the wrong size (about 1.5kb) ( , circled in red), AND sequencing reveals that plasmids from those colonies circled in red contain a fragment of Topo vector that should not be in there.

 

Running PCR on the purified plasmids from those colonies using T7 primers reveals similar results in that they contain a small mysterious fragment

I would truly appreciate any light you can shed on this matter. Thank you, and cheers!

-A

The 'faint secondary bands' look like non-specific amplification to me. I would start by getting rid of that non-specific amplification. Check your annealing temp (use the NEB Tm calculator). I think your annealing temp is too low. For Q5 the recommended annealing temp is Tm+3. You could also save yourself some time by reducing the time for the steps in your PCR. I would try the following:

 

 1. 98C initial melting temp, 2 minutes

 2. 98C melting temp, 10 seconds

 3. ___ annealing temp, 15 seconds

 4. 72C, 90 seconds (loop to step 2 34 times)

 5. 72C, 5 minutes (final extension)

 6. 4C, hold

 

 

I agree with Cutter about your cycling protocol. The long fragments you see on the initial gel are because your extension time is too long. You can eliminate them by reducing it. You probably need only about 45 seconds to amplify the 2.5 kb fragment. This is, however, not likely to be the problem. More likely you are trashing your DNA during the purification process.  Are you sure you even need to extract from a gel? Can you directly clone from the PCR mix (with a simple cleanup) after verifying you have the correct size band? The band looks strong and single in my viewing of the gel. I would purify the PCR product (Qiagen column or equivalent, or magnetic beads) then run a gel on the purified product to check that it was really there.

 

You haven't mentioned any of the other steps -- I just read the manual for the topo cloning kit, and they don't even require purification of the pcr product. Do less, get better results.  Make sure you really have a 5' CACC sequence on both primers, or the topo reaction will not occur.

Thanks to you both for the helpful feedback. To address your questions:

-About PCR: The first PCR gel is the result of using gene specific forward and reverse primers, and the gel shows clear, single bands at roughly the size I expected. It seems at first glance that the first PCR worked perfectly. Cutter mentioned some faint secondary bands, but all I see are faint bands that likely represent the plasmid from which I amplified. Annealing temperature aside, can excessive extension times somehow muddle the ends of my product? Until now, I presumed that excessive extension doesn't make a difference after first-strand synthesis

 

-About Purification: I assumed that I need to do some kind of purification because when I try to ligate directly from the PCR reaction, the insert does not go into the expression vector and I instead get colonies with the insert in the original sequencing vector.  This, however, could be due to problems with my PCR product as you both mentioned.  Is a PCR cleanup sufficient for retaining PCR product, but not retaining the leftover sequencing vector? Until now, I've been doing gel purification with a high concentration of DNA detected after eluting. However, I have even verified product on a gel.

 

Any additional feedback would be truly and greatly appreciated. I will also take your advice about changing my PCR parameters and let you know how it works out.

Many thanks, and cheers,

 

-A

Could you please explain this statement? Why should those be nonspecific bands and not the original plasmid?

Cutter on Fri Apr 1 20:35:48 2016 said:

The 'faint secondary bands' look like non-specific amplification to me. I would start by getting rid of that non-specific amplification.

A serious problem you have is that your template plasmid and your cloning plasmid apparently have the same antibiotic resistance. This makes selecting the correct clones very difficult. If possible, clone into a plasmid with a different resistance marker.

 

If you can't do that, then you can rediuce your background by using dramatically lower amounts of template (like a 1000x dilution) and increasing the number of pcr cycles. Then, add DpnI to your pcr product (no buffer etc. needed, just add 1 ul of DpnI) and incubate for 1 hour at 37. Then, heat kill the DpnI by incubating at 80 for 10 minutes. This selectively cuts the template plasmid (assuming it was purified from a lab strain of E. coli which was dam+ (most of them).. and leaves the pcr fragment uncut.

Thank you for the input.  I will try that and hope for the best, although I have to admit that I'm not sure why this method should work better than purifying the fragment (I always had good purification of the PCR fragment and verified it with a gel).  But I still could not find positive clones.

Therefore, it seems to me that the problem is more likely between my PCR fragment and the receiving vector, and not due to the same antibiotic resistance between vectors.  If you have any more light you can shed on this I would greatly appreciate it.

In any case, I will try your suggestion with DpnI and move forward.

Thanks,

 

-A

If you can see your plasmid template on the gel then you are using way too much plasmid template. You should use less than 1 ng of template. 

 

aodonnell311 on Fri Apr 15 08:52:37 2016 said:

 

Could you please explain this statement? Why should those be nonspecific bands and not the original plasmid?

Cutter on Fri Apr 1 20:35:48 2016 said:

The 'faint secondary bands' look like non-specific amplification to me. I would start by getting rid of that non-specific amplification.

 

Cutter on Sun Apr 17 09:26:06 2016 said:

 

If you can see your plasmid template on the gel then you are using way too much plasmid template. You should use less than 1 ng of template. 

 

aodonnell311 on Fri Apr 15 08:52:37 2016 said:

 

Could you please explain this statement? Why should those be nonspecific bands and not the original plasmid?

Cutter on Fri Apr 1 20:35:48 2016 said:

The 'faint secondary bands' look like non-specific amplification to me. I would start by getting rid of that non-specific amplification.

 

 

Thanks! I have added this to the things that I have tried in order to solve this problem. I have also changed annealing temperature and extension times.  I have also taken Phage's advice and digested the parental plasmid with DpnI.  I will report later on whether or not this worked to help others with similar problems...

 

Cheers,

 

-A