ITS gene PCR Smears - (Aug/31/2018 )

Hello Everyone,

As you can see in the picture for my PCR, I have very faint bands with surrounding dark smears. My positive control seems to be the darkest smear with no band present at all. My negative had no smearing. I will be sequencing my results. I would like to get suggestions please as to how I can get rid of the smearing and have more prominent bands. My samples are labelled 1 to 14. My negative control is - in red and the positive control is + in red.

I would appreciate all your ideas and suggestions to help me get through this.

Thank you so much!

Yazzy

What have you tried so far? 

I see that your negative does indeed contain some smear - throw out the reagents and get fresh ones in.

bob1 on Fri Aug 31 20:19:15 2018 said:

What have you tried so far? 

 

I see that your negative does indeed contain some smear - throw out the reagents and get fresh ones in.

Hello Bob, 

Thank you for your reply. I only tried playing around with the thermocycling conditions for example, decreasing the number of cycles, or decreasing the extension time. I also purchased new primers instead of my old primers. 

Yazzy

Please don’t try to sequence these samples, except maybe #14, after you run it through a PCR clean-up column.  In sequencing we have an expression: garbage in-garbage out.  You need good quality clean PCR products to get good sequence.   What size are you expecting?  Should they vary like that?

This gel is yelling “too much” but too much of what is the question.  Possibly your extension time is too long, or you have too much primer, or too much template, or too much Mg++. Maybe all of the above. What kind of template is it and how much did you use? Same with primers.

Hello Enthusiast,

Thank you for your feedback.

To be honest, I was not planning to sequence those products but I am hoping to improve the PCR to get better products for sequencing. I do expect variation in size because ITS gene is used for the identification of different nematodes.

The template is genomic DNA extracted from nematodes using the phenol with chloroform extraction method (I think since I don't do the DNA extraction myself). 

 I use 2uL in a 25uL PCR mix. Primers are 10uM and I use 0.5uL in a 25uL PCR mix.

Thank you again.  

Hi Yazzy,

Do you know how concentrated your template DNA is? For human genomic I would not put in more than 50 to maybe 100 ng in a 25 ul rxn, and I presume the nematode genome is smaller, so i'd put in 10-25 ng. It is possible that some of the smear you see is leftover template DNA. (For a plasmid you only need 100 picograms or so). Also, if there is any phenol-chloroform left over in the prep it can have a real negative effect on amplification.  Putting much less template in will help dilute out that contaminant.  (Column-based DNA extraction kits are recommended for PCR templates)

Your primer conc. seems to be standard (what kits usually recommend) but I got in the habit of using about half that much to get cleaner looking reactions. Try some experimental rxns with less primer and template, maybe half the amounts, a quarter of the amounts, and even 1/8 of the amounts, and see what you get. I hope it will help!

Thank you so much OldCloner. My concentration for my DNA is usually around 8ng/uL.

I will definitely try your suggestions and I will keep everyone updated about my results.

Regards,

Yazzy