DNA amount for RE reaction - (Jun/22/2015 )

Hi guys I need help.

I'm using NEB ecor1 and sal1 RE, my dna is a cloned ta vector plasmid, I wanna cut it and collect my insert.

I follow the manufacturer's protocol and only put in 1ug of the cloned plasmid in a 50ul reaction with 1ul of RE each.

When I cut the gel, I can see the band of the cut plasmid clearly, the cut insert was somehow vague or non existent.

So my question is can I increase the DNA concentration for RE above manufacturer's recommendation? Let say I put 4ug of DNA and thus I use 4ul of RE each in a 50ul reaction. Can I do this?

Thanks!

You can do this, but the efficiency of the reaction decreases with increasing DNA concentration. You are often better off doing multiple 1-2 ug tubes rather than increasing the DNA much more.

The reason you can't see your insert is because it is a fraction of the size of the plasmid: e.g. if you have a 500 bp insert and a 5 kbp vector and you digest 1 ug of vector, then the insert is only 100 ng (1/10th of 1 ug)...

bob1 on Tue Jun 23 08:47:40 2015 said:

You can do this, but the efficiency of the reaction decreases with increasing DNA concentration. You are often better off doing multiple 1-2 ug tubes rather than increasing the DNA much more.

 

The reason you can't see your insert is because it is a fraction of the size of the plasmid: e.g. if you have a 500 bp insert and a 5 kbp vector and you digest 1 ug of vector, then the insert is only 100 ng (1/10th of 1 ug)...

Thanks.

Upon calculation my insert dna should have more than 150ng, I'm surprised that I was not able to see the band in UV box. I wonder if there's mistake in measuring using nanodrop. Anyway, that's the reason why I am considering increase the dna concentration instead of doing multiple tubes, as I need to see visible band for cutting. 

Almost certainly your DNA is not as concentrated as you think.

In addition to thinking about the amount of DNA, you should also consider the VOLUME of DNA you add. Try to keep the volume less than 10% of the final volume. Otherwise, contaminant in your DNA preparation can cause problems in your digestions.

You should easily be able to see a band of 20-30 ng on an ethidium bromide stained gel.