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RFLP troubleshooting

RFLP troubleshooting - Molecular Biology Techniques

RFLP troubleshooting - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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  #1  
Old 05-04-2013, 02:55 PM
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Default RFLP troubleshooting



I have been carrying out Campylobacter flaRFLP for about a month. After I have amplified the target (flaA) by PCR, I cut the target with DdeI enzyme (Fermentas) for 8 hours according to the procedure suggested by Fermentas. I am using % 4 Nusieve GTG agarose and 100 bp ladder between 100-1000 bp during electrophoresis and rUn the digests at 90 V for 90 minutes. After staining with etidium broimde, the bands in the lower base pairs are faint. They are not as strong as the bands in the higher base pairs.

What should be the problem?
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Old 05-06-2013, 01:50 PM
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Default Re: RFLP troubleshooting

It always happens. I presume it has to be because of the size: shorter fragments appear faint because they get diffused on the gel more rapidly than larger fragments. Increasing gel concentration and decreasing electrophoresis times (as well as staining and visualizing ones) improves short fragments resolution. They will always look fainter than large fragments, though.

Another thing than you could try is to do small wells on the agarose gel to increase fragment signal: the smaller the band (in area not in base pairs) the strongest the signal, at least theorically.

Also, you could try polyacrylamide gels. They've got better resolution than agarose gels and are specially helpful in RFLP and other analyses involving fragment patterns.
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Old 05-07-2013, 03:46 PM
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Default Re: RFLP troubleshooting

Dear luisillo,
Thank you vey much for your tips. I will try to shorten my electrophoresis time as possible as the 100 bp marker let me do it.
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Old 05-08-2013, 01:31 PM
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Default Re: RFLP troubleshooting

What size are your fragments? Is there a considerable difference between the larger and the smaller? I ask you this because you should adjust the electrophoresis according to your fragment size more than to the 100 bp ladder.

You can also try to optimize staining times to ensure you get the maximum signal. For this I mean you stain the gel for a little longer and see if the lower bands look brighter. I advice you, you should take pictures of the gel before prolonging your usual staining time, just in case the gel spoils because of overstaining.

Post a pic of your gel if you can for further advice.

Good luck
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Old 05-08-2013, 07:46 PM
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Default Re: RFLP troubleshooting

Luisillo, my fragments are between 100 bp and 100 bp. I will do what you advice.

I post a pic. First lane is ladder. Second, third, fourth and fifth lane are different isolates. Please forget about the lane 8. Ladder is 100 bp (between 100-1000 bp). The last fragment in the first lane is 100 bp. I know tha ladder (first lane) was not good enough. I know that I have to move it furher in order to make it open appropriate. But since I want your comments about the fragments I am posting it. I can post a better pic in the future.

I wonder what would you say about this pic?
Thanks for your tips
Attached Images
File Type: jpg mehmet RFLP SON (2).jpg (67.9 KB, 5 views)
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Old 05-09-2013, 01:24 PM
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Default Re: RFLP troubleshooting

For what I see I don't think decreasing gel electrophoresis time should be wise. Actually you might as well increasing it a little in order to have better resolution of your ladder.

The gel looks very good. I can see what you say about smaller fragments (faint signal), except for the lowest, I presume it is an artifact like dimerized primers. You say your fragments are between 100-1000 bp but I'm seeing two bands below 100 bp in lanes 4 and 5, were you specting them?

As I said before, the gel looks good. I don't see any problem with your technique. The only thing I can tell you to do to improve further is to load more sample per well. Difference between large and small fragments will remain but at least you will have a brighter signal.

I hope this has help you. Good luck Gislab

Last edited by luisillo; 05-09-2013 at 01:25 PM.
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Old 05-09-2013, 04:10 PM
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Default Re: RFLP troubleshooting

Thank you very much for your help. As I said before, I am aware of the fact that the ladder needs to be run a bit further in order to see all the bands of ladder clearly. I will increase the electrophoresis time. As you said I will load more sample to the well.
By the way two bands below 100 bp in lanes 4 and 5 were also expected.
Thank you very much for your very important advices again luisillo
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  #8  
Old 05-10-2013, 02:02 PM
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Default Re: RFLP troubleshooting

Your welcome. Glad to be able to help you.

Good luck with your experiments gislab.
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