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Campylobacter RFLP troubleshooting

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Campylobacter RFLP troubleshooting

Postby gislab03 » Sat May 04, 2013 1:59 pm

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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Postby JackBean » Sat May 04, 2013 7:27 pm

Small bands are always fainter than big bands. What size are we talking about?
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Re: Campylobacter RFLP troubleshooting

Postby gislab03 » Sat May 04, 2013 9:27 pm

Dear JackBean, Thank you for your kind interest

My target amplicon is about 1700 bp. After restriction I have difficulty in evaluating the bands sspecially lower than 500 bp.
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Postby JackBean » Sun May 05, 2013 3:54 pm

Wait a sec. So you amplify your GOI and then cut it with RE for what exactly? Just at the ends to make sticky ends for consequent cloning? And I guess you use your PCR reaction without any purification?
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Postby gislab03 » Tue May 07, 2013 4:06 pm

I do not have any purpose of cloning.My aim is to type 300 Campylobacter strains molecularly and look for similarity between isolates; in short my aim is molcular typing. I am using RFLP just for that. Not for cloning or for something genetical method.
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