Dear Laura,

I think that’s normal when you run undigested plasmid due to the different conformations, as you mentioned. You can try linearizing your plasmid by performing a single digestion to see if you get the expected size. A double digestion would also help confirm whether both the plasmid and the insert are present, just to be extra sure.

I don’t think this indicates an issue with the cloning, especially since you're still getting the expected 1 kb amplicon. To be certain, you can send the PCR product for sequencing. I’ve encountered a similar issue before, but my sequencing results confirmed that the recombinant plasmid was indeed correct.

Hi Laura,

how much of the plasmid are using for your PCR?

For me it looks like you might have some of the Plasmid on your gel and your desired band of one kb looks very faint.

This can happen if you have to much template in your PCR. For a PCR on a plasmid 10 ng of template should be enough (and works usually better than to much template and you shouldn't see this on the gel).

For the rest I agree with Alif Ismail

Dear Laura,

The gel picture you show here looks like a ligated plasmid with more than a single insert (I will say 2 in your case). If I understand your question correctly, I think the much larger band of the plasmid (at 3Kbp in your case) after running colony PCR is very normal, as you use a colony as your template. In most cases, the next step would be sequencing to confirm your insert; however, in your case, with more than a single band of inserts, I think you need to confirm the double band first before proceeding. I mean, you can check your primer on a non-colony template to rule out double band or primer dimer as well. If that has been successfully ruled out, then you can take a step back and look at the colony process. Successful colony PCR of a ligated plasmid should show a single band of your insert.

Hi Laura,

I have seen that you also asked for the two bands you see with the undigested plasmid. I didn't mentioned it in my first reply, however your bands are indeed very likely due to the conformation of the plasmid. With the undigested plasmid you usually see 2-3 different bands.

Supercoiled DNA will run relativly fast - so this should be the lowest band on the gel.

Linearized DNA will run in the middle (however you will not always see this band or it will be faint - depending how much of your DNA is completly linearized).

Nicked DNA migrates slow in your gel - so this should be the highest running band.

I would assume that you see the nicked DNA and the supercoiled DNA on the gel with the undigested plasmid and this does not indicate any cloning issues.

To proceed you should first try the double digestion.

Use the the restriction enzymes you used for cloning, however if you have a double insert like Bimpe Suliyat Azeez suggested, you will not see this with this approach (in this case at least one of the restriction enzymes should cut the insert exactly in the middle and you will only see one band).

To see if you have a double insert you can digest the plasmid with restriction enzymes flanking the original restriction sites ( I am not sure if this is the problem, however it is a possibility). I would do both approaches.

If you also want to confirm the PCR you can do this in parallel (with less DNA). With this approach you should be able to identify your positive colonies relativly fast.

KR Dörthe

Bimpe Suliyat Azeez when I perormed the colony PCR I only had one band. The 3000 bp appeared after the PCR performed on the plasmid extracted with the mini-prep; that's what left me very confused. I used more or less 50 ng of plasmid for the PCR.

Dörthe Andrea Kesper thank you for your help. I am currently sequencing my plasmid, so I will let you know how was it!

Please do share the result here Laura Dionisi

Bimpe Suliyat Azeez I know that the PLASMID PCR photo is not very clear, I realised I missed the photo of the gel at the end of the run, but still a higher band than expected was observed (it was higher than the 2000 bp marker).

The size is higher than your expected size? Is that what you mean? Laura Dionisi

Hi Laura,

I hope you get the your results after sequencing the plasmid.

Howeve,r I had a look at your PCR results and for me it seems that you now have a band at same position were you had the very faint band in your first gel picture from which I understood that this was your 1 kb band. But as I don't know which marker you are useing this is hard to tell. Is the strong band that you are seeing know higher than 2 kb?

You might give me some more information about your PCR conditions (like what polymerase did you use, primer concentration, annealing temperature, elongation time e.g.) to work on the optimization of the PCR. For me it seems that you get also some small additional PCR products and/or primer dimers. I would still considering doing a digestion with restriction enzymes. However, before you work longer on the optimization of your PCR or on a restriction analysis it might be better to wait for the sequencing result. I would be really interested to know what your results are.

KR Dörthe

Bimpe Suliyat Azeez yes, I was expecting a 1000 bp product. My marker first band is 2000 bp, and at the end of the electrophoresis I saw one band at 1000 bp, and another faint band which was higher than the 2000 bp marker band (the first one you see in my picture)

Dörthe Andrea Kesper Bimpe Suliyat Azeez

I think I wasn’t clear in explaining what can be observed in the gel.

In the picture of the PCR on the plasmid there are 3 wells:

1) marker

2) PCR product

3) negative control

In the middle of the lanes (2 and 3), there is a band corresponding to my PCR product (1000 bp). Please note that the image attached is not from the final stage of the electrophoretic run—I unfortunately lost that one. As a result, in this picture the PCR band appears slightly higher than expected, roughly near the 2000 bp marker. However, its actual size is 1000 bp.

Additionally, a thin band is visible above that, which—by the end of the run—was found to be larger than 2000 bp.

Dörthe Andrea Kesper

I used a Qiagen HotStart Taq DNA polymerase, with primers at a final concentration of 0.2 µM in the PCR mix. I added 5 µL of plasmid DNA to a final volume of 25 µL (20 µL PCR mix + 5 µL DNA, concentrated about 20 ng/µL). The thermal cycling protocol was as follows:

• 3 minutes at 94 °C (initial denaturation)
• 35 cycles of: → 1 minute at 94 °C → 1 minute at 63 °C → 1 minute and 15 seconds at 72 °C
• 10 minutes at 72 °C (final extension)

The annealing temperature I chose is slightly low (the calculated annealing temperature is actually a few degrees higher); I might consider increasing it to raise stringency and potentially reduce non-specific products. In any case, I'm still waiting for the sequencing results before deciding how to proceed.

Hi Laura,

I hope there was a typo, because in lane three (negative control) should be no band ( and luckely I don't see one). If I see it corecctly your colony PCRs are all positive except the one in lane 8. The high running band might be still plasmid DNA as you are still using a lot of template (and it iseems to be not present in the negative control without template).

If you doing PCR on a plasmid a total of 10 ng in your reaction should be enough - you are using 100 ng in your reaction at the moment. (Thermofisher recommeds 0,1-1 ng in a 50 µl reaction - so 10 ng is still a lot).

Often people are hesitant to use less template but if you still want to improve your PCR results please try it.

Some additional tips for improving your PCR:

1. Your extension time might be to long - the extension rate of the Quiagen Hot Start DNA polymerase is 2–4 kb/min at 72°C (according to Quiagen). (30 sec should be enough with this extension rate).

2.Optimize your annealing temperature - if possible do a gradient PCR

3. Your annealing time might be also a bit to long. Long annealing times can lead to primer dimer formation (and it seems like you have primer dimers) and non-specific primer binding (try 30 sec).

Please let me know your sequencing results - I am really interested :-)

KR Dörthe