11 replies to this topic

[shivu11]

Dear All,

I am trying to clone a 900bp gene. I have amplified the gene from genomic dna. and i have added NDE1 and XHO1 sites. I have been trying to clone this gene since 6 months, but no success. I do digestion for 3hrs then gel extract it and do ligation for 16hrs at 15degree. Then transform it in DH5a, I get nice colonies after that. I isolate plasmid from 2-3 colonies and do RE digestion and PCR for confirmation. In PCR I get number of bands, inculding one at 900 and others at 3000, 2000bp. and when I do digestion I get two bands, one at 5000( vector) and one between 2500 and 3000bp. I dont knw what is happening. Please help. I have to clone this gene.
Thanks in advance.

[Pangea]

Q5: It seems that NdeI is having some difficulties cutting my DNA. Is there a reason for that?A5: NdeI activity is sensitive to contaminants present in DNA isolated with standard miniprep protocols. Further purification of the DNA by a column or dialyzing the contaminant from the DNA may help increase the activity of the enzyme on the isolated DNA substrate.Q6: NdeI seems to be digesting my DNA correctly, but when I try to ligate it, I obtain no colonies. Is there a potential explanation for what I am observing?A6: NdeI is a very robust enzyme. If the substrate DNA is digested for extended periods of time, we have found evidence that the enzyme will remove some additional nucleotides. Digestion of DNA with NdeI over 4 hrs is not recommended.

Maybe your gene is multiple cloned into the vector.And using 2 or  3 colonies is pretty few. And your primer might be not specific enought.

[shivu11]

Thanks Pangea, How we can prevent gene to get multiple cloned into the vector ? any suggestions will be very useful !!

[Pangea]

It might be that your digestion of your Insert is not working out you have blunt ends. So it will just ligated several inserts. But i am not sure. And i hope you are not using BL21 for plasmid replication.  Not recA mutation. But BLR have it or other K 12 strains. And are sure that you are not cutting into your plasmid?

Edited by Pangea, 05 January 2013 - 01:23 AM.

[leelee]

Can you post up a gel pic of both your PCR and your digests?

It is worth considering that the unwanted bands you are seeing is uncut plasmid.

How exactly do you do your PCR from your clones? What is your template (e.g. scrape of colony in water, or mini-prep), and how much are you using for your PCR?

[shivu11]

Pangea, on 05 January 2013 - 01:18 AM, said:

It might be that your digestion of your Insert is not working out you have blunt ends. So it will just ligated several inserts. But i am not sure. And i hope you are not using BL21 for plasmid replication.  Not recA mutation. But BLR have it or other K 12 strains. And are sure that you are not cutting into your plasmid?

thanks pangea for the reply, I use DH5alpha for transformation, I m sorry but I didnt get what u meant by cutting into plasmid.

[shivu11]

leelee, on 06 January 2013 - 12:16 AM, said:

Can you post up a gel pic of both your PCR and your digests?

It is worth considering that the unwanted bands you are seeing is uncut plasmid.

How exactly do you do your PCR from your clones? What is your template (e.g. scrape of colony in water, or mini-prep), and how much are you using for your PCR?

DearLeelee, I isolate plasmid and then use it as template, about 0.5microl litre is suffecient. I isolate plasmid using mini prep kits.

[leelee]

shivu11, on 06 January 2013 - 01:52 AM, said:

leelee, on 06 January 2013 - 12:16 AM, said:

Can you post up a gel pic of both your PCR and your digests?

It is worth considering that the unwanted bands you are seeing is uncut plasmid.

How exactly do you do your PCR from your clones? What is your template (e.g. scrape of colony in water, or mini-prep), and how much are you using for your PCR?

I would suggest the following steps:

1. Dilute your template for PCR 1 in 10, 1 in 100 and 1 in 1000. Do a PCR reaction with each of these. Do the unexpected bands still appear?

2. Use less template when you perform your digests for screening, and make sure that you are digesting in a sufficient volume compared to the volume of template you are using (you ideally want your template to make up 10% or less of your reaction volume, especially if you have eluted your DNA into a buffer rather than water).

And when running the gels for both the above, run an uncut plasmid lane too.

DearLeelee, I isolate plasmid and then use it as template, about 0.5microl litre is suffecient. I isolate plasmid using mini prep kits.

0.5ul from what amount? And extracted from what amount of culture. High copy number or low?
This doesn't really tell me much. Did you quantify the DNA or estimate your yield from a gel?

Have you run uncut plasmid next to your digests when screening your clones? If so, are the unwanted bands in your digest running to the same size as your uncut control?

[Pangea]

You have right you have single cut RE. Sorry about confusing.

[shivu11]

I do not estimate the dna conc. We usually isolate plasmid from 5ml culture, inoculated overnight. Gel shows gud intensity band. and my plasmid size is 5000bp. and band which I am getting is at 3000-2500bp.
Thanks again

[leelee]

shivu11, on 07 January 2013 - 01:11 AM, said:

I do not estimate the dna conc. We usually isolate plasmid from 5ml culture, inoculated overnight. Gel shows gud intensity band. and my plasmid size is 5000bp. and band which I am getting is at 3000-2500bp.
Thanks again

Well, you will likely have a bucket load of DNA from a 5ml mini prep, so using that as neat template for PCR may be your problem. Have you considered trying the dilutions like I suggested?

Are you open to the possibility that this 2500-3000bp band you are seeing is uncut plasmid? Or have you dismissed that idea, or ruled it out someway or other?

[shivu11]

thanks Lelee,

thanks for the reply. I am trying to clone the gene again, and hope to get the success this time, Thanks again to all for ur time and patience.