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wrong size in agarose gel, right size in sequencing

wrong size agarose sequencing

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7 replies to this topic

#1 cleopeptid

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Posted 02 September 2011 - 06:31 AM

Hey,

I made colony PCR and applied the samples on agarose gel (1 %, TAE 1x, EtBr in the gel, 30 min, 90 V)
Colony_PCR.jpg --> the picture of my gel

I used GeneRuler 1 kb, I used the same aliquote for all my other runs and always the correct bands were next to the correct ladder band in the gel.
This time I received (as you can see) 750 bp for all bands (the ones without bands are negative controls). But I expected 1100-1200 bp.
I didn't sequencing of three colonies and they had the correct insert with the expected size.

Now what went wrong?

- the GeneRuler worked fine afterwards, too.
- a problem by running two gels in one, but another colony PCR made the same way with another fragment (one week later) worked fine?

Thanks for suggestions.

#2 Ameya P

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Posted 02 September 2011 - 10:29 PM

Cleo,

Please check the sequence of your insert for REstriction sites once again. Probably you missed the one more site of the enzyme you are using, which is why you are getting the smaller band.

Also, when you sequencing three colonies. They had the correct insert sequence. Could you tell us, what was the size of the sequence read? Did you get the whole insert or did you just randomly check for the initial few bases ?

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#3 cleopeptid

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Posted 02 September 2011 - 11:25 PM

Hey,

I sequenced the whole insert with the 1100 and 1200 bp. So it is correct, just the gel doesn't fit.
I checked the seuquence again for my restriction enzymes and their are no other sites. It is a fusion protein of my protein and ECFP.

#4 Ameya P

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Posted 03 September 2011 - 04:14 AM

Well, if your sequence is fine, then thats good news. Regarding the picture, can your repeat the PCR and see if it gives the same results. also, please check the PCR program you had used for this run. Could it be possible that the program was edited and you did not know about it?

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#5 cleopeptid

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Posted 03 September 2011 - 04:40 AM

Hey,

no I cannot repeat the PCR, as it was a colony PCR. I'm writing my master thesis and I just saw that these are the wrong bands when I added the bp of the ladder. Now I was wondering why, because the sequence is fine, and the PCR was working all the time.

#6 bob1

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Posted 03 September 2011 - 04:24 PM

Could it be that there is an alternate binding site for one of the primers in your sequence?

#7 cleopeptid

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Posted 04 September 2011 - 07:03 AM

I just checked the sequence and there is no other anneling site for the primers, even for fragments of the primers (it is the pVITRO2 primer forward and reverse).

#8 Ameya P

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Posted 04 September 2011 - 09:23 PM

Probably the ladder you loaded is not Gene Ruler 1 kb. Only then it cam make sense. Ask around if there are any other ladders that are used in the lab.

NEW!!!!  RotM: Interview with Prof. Kenro Kusumi on CoffeeTableScience!!!!

Image copyright: Adrian Koh SF.
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