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contamination PCR


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#1 ZZCC

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Posted 17 December 2003 - 03:42 AM

...can you help me ...just with explanation...

I sent my samples for sequencing and I have got results...but that confuses me very very hard...that is my first PCR experiment so I would like to know where I made a mistake...

I had lactobacillus template ...and I used protocol for lactobacillus amplification...with primers P1V1, P4V3...and I send all of that to MWG and when I have got the sequence after putting that in BLAST program on NCBI site I had result Acinetobacter...?????

Ok I know that I can have some contamination but Acinetobacter was ever never found in my lab...

And when I have a picture from gel after PCR reaction (with lactobacillus and primers like I sad before) I have got band (one small...not important to me...which can be some contamination)...and another band on cca 700 bp...which have to be my lactobacillus...(it is in literature that lab with that primers gives product about 700 bp...correctly 697 bp)...

What really happends...to me...???

#2 labrat

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Posted 17 December 2003 - 09:59 AM

I have two questions:

Is your sequencing result looks good, how many reads?
How much homology to Acinetobacter did you get by blasting? Did your sequence has any homology to your desired lactobacillus? and how much homology between these two organisms?

#3 ZZCC

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Posted 17 December 2003 - 10:15 AM

YOU ASKED
Is your sequencing result looks good, how many reads?
How much homology to Acinetobacter did you get by blasting? Did your sequence has any homology to your desired lactobacillus? and how much homology between these two organisms?



what means ...my sequencing results good..?? I have 4 results of sequencing and beetwen them is almost excelent homology..
I did comparation between 16S rRNA Acetinobacter and Lactobacillus and homology is cca 65%...the primers I used are not specific for Lactobacillus the give me that protocol and how I am new I think that is the low...

when I look at the picture of gel after electrophoresis my PRC product which I had sent to sequencing I have two bands...first at about 300 bp and the second about 700 bp...(that 700 is my desired product)...and I supose that 300 is some contamination...

primers has almost the same way of aniling to both DNA...acetinobacter and lactobacilus...that I undestand latter

is it posible that even I send my primer to MWG they sequenced that first product and dont take care of the second (700 bp) because i didnt tell them what I really need and I didnt tell them what happends and all of that because I didnt think that that primers I uset to amplifie Lactobacilus DNA can be used for amplification of Acinetobacter also

#4 labrat

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Posted 17 December 2003 - 12:20 PM

It seems to me that you sent your pcr product (which had two bands and they had not been separated) for sequencing. You think the 700bp band is the product you expected. I would suggest that you only sequence the right band by cutting it from your gel and purifying it.




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