Hello Elyn,

There are a couple of things I want to know:

Is there a particular reason that instead of using the plasmid, you are using an eluted and digested fragment? Because if you are only interested in the PCR fragment of whatever gene, you can usually use the entire plasmid to do the PCR.

Secondly, from your gel profile, it does seem that there is still some plasmid left, the upper bands are from the presence of too much of either linearized or undigested plasmids. It would be good to have the negative no DNA control in the gel as well as you can add the template you used for the PCR in this gel.

Third point, If you only need the PCR product, then your PCR product is good in terms of the size of 237 bp as you mentioned. If this is the only band you are interested in and you do not want to go into troubleshooting and getting a perfect single band PCR, then you can further elute this product and use it for downstream work. The upper bands should not matter.

Finally, RE are used as 1 unit/microgram of DNA, so "the suggested plasmid/RE ratio is plasmid: RE = 1:1( 1ug for 1ul enzyme).the amount of RE that I usually use is 2ug" is not correct way to put it. One microlitre of enzyme can have 5 units to 20 units depending on your enzyme and company. But it looks like you are using lot of enzyme and EcoRI has star activity, that means that when the enzyme is in higher amounts than suggested by the manufacturer, then it can start cleaving sequences that are similar though not identical to its defined recognizing site. And as you say, there is no problem in gel and you see the digested product. So, this should not be a problem, but I wanted to add this point FYI.

Best wishes,

Simran

One restriction enzyme unit is the amount of enzyme that will cleave 1 ug of DNA within on hour. That doesn't mean that if you have 1 ug of DNA, you should use 1 U of enzyme. Load that reaction up with more restriction enzyme and guarantee that you are cleaving your plasmid!

Additionally, PCR is a great linearizing technique. Just as simran said, you don't even need to digest the plasmid before doing the PCR. Just make a PCR reaction and add between 5 and 50 ng of plasmid DNA into a 100 uL PCR reaction. If your PCR is successful, you will have a significant amount of linearized product and barely any contaminating plasmid template. 

I would almost guarantee that you are using way too much template in your PCR reactions. Polymerases are really awesome, but they are all slightly different. You should use this chart to try and understand each one's characteristics (https://www.neb.com/tools-and-resources/selection-charts/dna-polymerase-selection-chart). Taq DNA polymerase, for example, has a DNA binding Kd of 2 nM. As long as your starting template concentration is within 2 nM the PCR will work really well. 

Agree with all... Is that genomic DNA?

Thank you very much for all the feedback.

I actually want to do sox9 sequencing. That is why I cut and run PCR for the digested plasmid. I am also doing it on the whole plasmid.

A minor typo error, it is actually 2ug of plasmid, not 2ug of RE.

Anyway, I will try all the suggested methods. Thank you again!

Hello again Elyn,

Even for sequencing, you do not need to do the PCR, send the sequencing company the plasmid and the primers you are using for amplification. And if it is some common plasmid, like pUC series or some other, then you can even ask the company to use their own commercial primers for sequencing.

Best

Simran

Hello Elyn,

If your aim is sequencing it is better to do it directly on the plasmid, as Simran suggests. Moreover, the more steps you do the more errors you may get in the sequencing. Specially if you PCR without using a proofreading taq!