Colony PCR can be very tricky when it comes to reliability of the result. While you are plating the transformed bacteria, you are also spreading the untransformed remaining DNA  on the plate. That's why while you are trying to pick the colony, you can also obtain this untransformed DNA in your template solution. When you make PCR with that even if the untransfomed DNA amount is little in your colony suspension, it still gives band. In this case I recommend you to do a PCR also on your miniprep product, not directly to the colony. Good luck!

What's the size of your insertion?  Did you find the plasmid exacted from your ligation larger than the void vector? If not, I would consider double digestion results more reliable. The false positive rate is quite high in colony PCR because there is remaining ligation in the solution you scribble in the plate probably. 

size of insert is 1.5kb. yes. yes I double digested it but was not my expected size.

What's the size of the produced band? If it doesn't differ a lot with your prediction, you can send several colonies for sequencing. 

they differed alot, My expected band was 5.3kb and 1.5kb. But what i got is single band at 10kb

Then I will assume that only single digestion happened. Have positive colonies sequenced . It won't cost a lot, both money and time. 

NdeI is a tricky enzyme, I would suggest to use a higher fold of enzyme and longer time of incubation. You can also try digesting with NdeI overnight and later with XhoI for 3 hr and gel the digest on the gel.

Colony PCR can be very tricky when it comes to reliability of the result. While you are plating the transformed bacteria, you are also spreading the untransformed remaining DNA  on the plate. That's why while you are trying to pick the colony, you can also obtain this untransformed DNA in your template solution. When you make PCR with that even if the untransfomed DNA amount is little in your colony suspension, it still gives band. In this case I recommend you to do a PCR also on your miniprep product, not directly to the colony. Good luck!

You pick your colony at day 2. It is a problem : the stability of your antibiotic is only around 24H. If you pick at day 2, may have some satellite colony that not have your plasmid. In addition, when your culture is too advance you may have some modification of your plasmid by the bacteria due to high density. With DH5a, 24H of culture is enough.

Hey Lelamekala,

To ensure that your restriction conditions are appropriate, I would suggest to run a control digestion of the parental plasmid with each enzyme individually. Whenever the digestion works, you will only see a single DNA-band in electrophoresis, the linearized vector.

Just that I understand it right: When you state that after digestion, you "you elute out" single bands from vector and insert, does this mean that you seperate them by gel electrophoresis?

Instead of sequencing, you could also consider to use a restriction enzyme somewhere in the plasmid (i.e. not the ones you use for cloning) to check whether your plasmid is there. 10 kb after digestion is not very promising that the cloning worked, I assume...

Anyway, maybe it was just bad luck this time!!

Best regards,

Christian

NdeI is extremely sensitive to impurities, especially salts. So failure to digest could reflect a problem with the quality of your miniprep. It is a good idea to sequence the plasmid if it is larger than empty vector, but in this case, it is much too large (an extra 3.5kb) to be consistent with a single insertion event, and may indicate a contaminating plasmid. Was the template DNA used for PCR (not the colony PCR) of your insert also a KanR plasmid? If so, maybe you simply recovered the source plasmid for your insert rather than pET26.

Thanks everyone.

Kerya - Yes, I did my PCR by using my miniprep. U got few multiple bands ( with 3 thick one) -which one of it is my expected band. (attached pic)

Christian - Yes, I run all of the in a single gel (attached pic). Yes I elute out by using gel extraction method.And yes I tried with BpiI to check if I'm working with correct plasmid.

Daniel - Nop,my insert was from chloramphenicol resistant.

attached is my pcr product from miniprep. ( three distinct bands) 1kb bioron ladder

the last four lanes are my digestion. 1) nde1 (5360bp) 2)xhoi1(5360bp) 3)ndeI and xhoiI (5230bp+130) 4)digest with bpiI and that was the expected size (4219bp+339+388+360)

Ladder (1kb Bioron) all was at expected size

*the two gels are same.one was run longer and one shorted.

You should try some specificity controls for your colony PCR- for example, no template negative control, and/or a colony from a different transformation that wouldn't have your insert. This will help you interpret whether you are really detecting your cloning insert or not.

I do have control for my colony pcr. I add 1ul of H2O.

Try screening more colonies. Personally, I have never observed problems with false positives in colony PCR due to contaminating DNA from the ligation reaction, so I'm a bit skeptical of this explanation. Nonetheless, it is a good idea to miniprep plasmid from 3 or 4 different colonies, in case some don't behave in the expected manner in the restriction digest.

Salam friend, colony pcr we usually use it for confirming the presence of virus using specific primers before going to miniprep step ( after transformation in plate)

and typically the digestion of the sample you will do it to confirm the full virus inversely to colony pcr which focus mainly specific region of the genome

Thanks

It can be happened. You should screen more colony to find out our real construct.

screened more than 10 colonies. All shows neg result. :(

DNA contamination while doing colony PCR protocol may lead to false positive results. If template DNA gets into reaction mixture by mistake(while handling), it can show amplification bands on gel. Take precautions while doing it.

Checking by Restriction digestion can be the ultimate result.

If screening of more than 20+ colonies is giving out negative results, then probably your insert is not with e.coli. Recheck your ligation step. Comp cell efficiency. etc. 

There can be many factors responsible for why your gene is not ligating with the vector.

Ok. Thanks for all the replies. Looks like I have to redo the whole experiments. 

I think you should carefully do the ligation step.

When you set up your ligations, you need to include a no insert negative control. This will allow you to assess the background rate of colony formation due to vector alone. This should be much lower than with your insert. Little hope of success if you can't see a difference between the number of colonies with vs. without insert.

Ok.The concentration of my vector ( 15.3ng/ul) and insert (101ng/ul). I will be using 1:3 ratio for my ligation. Vector 50ng and insert 150ng. Is it okay?

No! You need to calculate based on molar ratio, not total mass. For a 3:1 insert:vector, you would need 3*50*1500/5360, or about 42ng of insert per 50ng of vector. Best not to exceed 100ng of total DNA in your ligations. So, if you wanted to conserve vector, you could set up your ligation with 25ng of vector and 21ng of insert.

tried all ways and i still didnt get any colony... going to be 2 months ready :( please help me

Have you checked the transformation efficiency of your competent cells using a positive control? 10^8 CFU per ug DNA or better is preferred. Also, take care that you do not overexpose your DNA to UV during gel isolation. This can damage your plasmid by causing adduct that will not allow replication of the plasmid, and thus impede your cloning.

I'm using NEB DH5a competent cells (commercially purchased) which is apparently working well for everyone. Ok, I will try to mininize to UV light but was not very concern about UV so far. Thank Cohen.

How far are your restriction sites from the ends of your PCR product? Most enzymes need 3-4 bp of flanking sequence to cleave efficiently. Have you tried sequencing your vector to make sure the MCS matches your expectation?

How do you obtain your insert is crucial if you have PCR amplified and then restriction digested then it is ideal to check for a Taq polymerase of better proof readingt activity and then PCR amplify. Further try cloning in another vector of same restriction site and see whether cloning occurs.

Hi,

I have similar problems, did you get the correct construct finally, if how, how? 

thanks.

Hello Nagabushan,

Yes. I have successfully cloned the gene into pet26. Apparently the previous positive PCR result has nothing to do with the transformation success. I re-digest, re-ligate and re-transform them and it worked.

Thanks everyone for the help and advice. :)

I have a similar problem as Lalamekala. The colony pcr showed was positive but the restriction digest was not. 

Vector: tagRFP-N : 4.7 kb

insert: ABCC2 : 4.6 kb

1:3 molar ratio 50ng vector and 150ng insert, total DNA of 200ng

I get about 20 colonies and screen  about 10.

Less than 10s exposure to DNA to UV.

i have repeated this several times back and forth trying molar ratios of 1:4 and even 1:9. Please help.

Lalamekala, do you mind sharing what conditions worked for you eventually?

Daniel any ideas for my specific situation?

anyone to help?

Thanks 

Thank you everyone for your kind contribution. Recently I faced similar situation, and would like to share it, so that someone else would be benefited.

Vector : pet16b

Insert : 1308 bp

RE : NdeI and XhoI

Following the double digestion and ligation, I got many colonies and my colony PCR (with Gene-specific primers) indicated 4 colonies as positive out of 10. I sequenced all of them and failed.

After having read the discussions, I repeated the PCR with purified plasmid of these 4 colonies individually with

1)     Pair of gene-specific primers

2)     Pair of vector-specific primers

I got the false positive (! As already sequencing is failed) data for PCR with gene-specific primers, but vector-specific primers did not show any bands, implying reliability of using vector –specific primers (Fig. 1).

Next, I picked further colonies from the same plate, and along with the plasmid I already ad purified, I performed the PCR with one gene specific forward primer and one vector-specific-reverse primer. Data showed no amplification (Fig. 2).

Based on this, I suggest that combination of gene and vector specific primers is best to screen colonies, to avoid unnecessary confusion.