10 replies to this topic

[libra84]

Hi everyone, I need some help with cloning a 1.5kb DNA fragment into the 4.9kb pGEX-3x plasmid.

First, i have designed primers to amplify the DNA which is already in another plasmid and at the same time added the EcoRI and BamHI restriction sites flanking the DNA fragment. I have also included 3 to 4 additional bases in front of the restriction sites. After PCR, i cut the desired DNA band and performed gel extraction using Invitrogen's Purelink Gel Extraction kit. After that, i used Nanodrop and the DNA concentration usually ranged between 6 to 9 ng/ul.

For the plasmid, I transformed it into Top10 cells and later mini-prep to get more of the plasmid. Nanodrop reading showed around 1600 ng/ul.

Then, i digested 5 ug  of the plasmid in a total volume of 50 ul. For the DNA fragment, all 30 ul of it obtained from gel extraction was digested in a total volume of 40 ul. EcoRI from Promega and BamHI from NEB was used for the double digestion. Buffer used was NEB buffer 3 and BSA. Reaction was performed at 37C for 1h. So after the 1h, i took 5 ul of the reaction mixture and run it on agarose gel. No bands were seen and i dunno whats wrong... I have done this twice and the same results.

Any help would be greatly appreciated. Thanks!

[chimpsarehungry]

Have you done single digestions of each EcoRI and BamHI to check for plasmid linearization to see if the enzymes are working?

[libra84]

chimpsarehungry, on 05 April 2011 - 08:17 AM, said:

Have you done single digestions of each EcoRI and BamHI to check for plasmid linearization to see if the enzymes are working?

Hi

I have not done this. I reckon i do not need to do this as someone else in the lab has used the EcoRI with success in his cloning and the BamHI was bought less than 1 month ago. Do you think i make sense?

[phage434]

This checks whether your restriction sites are present on the plasmid (and that they aren't methylated accidentally, e.g.).  The enzymes likely are working, as you suggest.
Also, that is a very high concentration of DNA.  Make sure you are doing reactions where only a small fraction of the final volume is from the DNA.  Otherwise, common inhibitors present in the DNA can prevent digestion.  I'd recommend digesting only 1 ug of DNA in a volume of 50 ul, with most of the volume being water.  Are you sure buffer 3 is the correct buffer?

[libra84]

phage434, on 05 April 2011 - 07:52 PM, said:

This checks whether your restriction sites are present on the plasmid (and that they aren't methylated accidentally, e.g.).  The enzymes likely are working, as you suggest.
Also, that is a very high concentration of DNA.  Make sure you are doing reactions where only a small fraction of the final volume is from the DNA.  Otherwise, common inhibitors present in the DNA can prevent digestion.  I'd recommend digesting only 1 ug of DNA in a volume of 50 ul, with most of the volume being water.  Are you sure buffer 3 is the correct buffer?

I see. But the EcoRI and BamHI sites are only separated by less than 10 bases. So even if it works, I wouldn't be able to tell from the gel am i right?

Anyway, NEB recommends using Buffer EcoRI for this double digestion. However, as my EcoRI is from Promega, i do not have that buffer. In addition, the NEB chart shows that EcoRI works in all buffers while BamHI works best in Buffer 3. Hence i decide on using Buffer 3.

Actually could the problem be due to the digestion? Even if the enzymes don't work, i should still get bands on the gel right? It seems that the DNA is gone and i don't know why that is happening.

[libra84]

Just prepared new autoclaved Milli-Q water and newly diluted BSA for the digestion. Repeated the experiment with no bands again after digestion....

Should i just continue with the cloning despite having no bands?

[Shiny]

When you say there are no bands, do you mean the ones you expect to see after digestion or there are not any bands as in the gel is empty. Restriction enzymes are sensitive to contamination and also because your sites are just 10bases apart its a good idea to run single digests of the vector with BamHI and EcoRI to ascertain that the enzymes are working.
If there are not any bands at  all, this suggests possible contamination.

[libra84]

Shiny, on 06 April 2011 - 10:59 PM, said:

When you say there are no bands, do you mean the ones you expect to see after digestion or there are not any bands as in the gel is empty. Restriction enzymes are sensitive to contamination and also because your sites are just 10bases apart its a good idea to run single digests of the vector with BamHI and EcoRI to ascertain that the enzymes are working.
If there are not any bands at  all, this suggests possible contamination.

Oh i mean the gel is empty. So you mean the enzymes are contaminated with DNase? Which results in no DNA after digestion?

[dedee]

libra84, on 06 April 2011 - 11:23 PM, said:

Shiny, on 06 April 2011 - 10:59 PM, said:

When you say there are no bands, do you mean the ones you expect to see after digestion or there are not any bands as in the gel is empty. Restriction enzymes are sensitive to contamination and also because your sites are just 10bases apart its a good idea to run single digests of the vector with BamHI and EcoRI to ascertain that the enzymes are working.
If there are not any bands at  all, this suggests possible contamination.

Oh i mean the gel is empty. So you mean the enzymes are contaminated with DNase? Which results in no DNA after digestion?

You could try to do a PCR screening. That doesn't solve the problem with the restriction enzymes, but you know whether your insert is there. Careful with using the exact same primers you use for cloning though - this can give false positives.

[libra84]

dedee, on 07 April 2011 - 03:39 AM, said:

libra84, on 06 April 2011 - 11:23 PM, said:

Shiny, on 06 April 2011 - 10:59 PM, said:

When you say there are no bands, do you mean the ones you expect to see after digestion or there are not any bands as in the gel is empty. Restriction enzymes are sensitive to contamination and also because your sites are just 10bases apart its a good idea to run single digests of the vector with BamHI and EcoRI to ascertain that the enzymes are working.
If there are not any bands at  all, this suggests possible contamination.

Oh i mean the gel is empty. So you mean the enzymes are contaminated with DNase? Which results in no DNA after digestion?

You could try to do a PCR screening. That doesn't solve the problem with the restriction enzymes, but you know whether your insert is there. Careful with using the exact same primers you use for cloning though - this can give false positives.

What do you mean by PCR screening? N i don't even know if there is a problem with the restriction enzymes. They should be fine since they are new and one is used by others in the lab.

[Shiny]

to test if your enzymes are contaminated or the DNA is you could use another DNA prep or even lamda DNA which has similar restriction sites and trying digesting them. If you see smear with that that means the enzymes are contaminated. If you see it only in your DNA prep that means the DNA is contaminated. Best way would be to prepare a new preparation or try to phenol chloroform the old one and see if you get any bands.