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isolation of plasmid (midi and maxi prep)


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18 replies to this topic

#16 ascacioc

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Posted 29 August 2012 - 03:03 AM

Even if you cut at 1013, it does not matter: it is a circle that you cut at one position; no matter where this position is, the result is the same : one band of the total length of the circles circumference.

Now that I have seen your map: do not use SfiI for cloning; if you cut with SfiI from the GeneArt vector, you will have your insert but you will not be able to control the direction of insertion. So, use this plasmid from GeneArt as a template for your PCR and insert EcoRI site on the 5' end and XhoI or NotI at the 3'. Again: take care that your gene will be in frame with the rest of the tags, VERY-VERY IMPORTANT!!

Andreea

#17 siddharthsameer

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Posted 29 August 2012 - 06:22 AM

Even if you cut at 1013, it does not matter: it is a circle that you cut at one position; no matter where this position is, the result is the same : one band of the total length of the circles circumference.

Now that I have seen your map: do not use SfiI for cloning; if you cut with SfiI from the GeneArt vector, you will have your insert but you will not be able to control the direction of insertion. So, use this plasmid from GeneArt as a template for your PCR and insert EcoRI site on the 5' end and XhoI or NotI at the 3'. Again: take care that your gene will be in frame with the rest of the tags, VERY-VERY IMPORTANT!!

Andreea

THANK You so much, now i am releved and now i can perform my rest of the experiment.....You are a saviour.. vielen dank :), any how i did my pcr today and tomorrow i will check on agarose... hope everythng is fine...and will let you know :)

#18 ascacioc

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Posted 30 August 2012 - 02:39 AM

I will post here what Sameer has written to me on the personal conversation and also try to answer it:
yesterday I did the pcr using my sequence that i had it from midi prep but Today wzhen i saw it it had different band of 2 kb but where i expected it to be of 1,4 kb.., i mad pcr of 4 samples that all had the same.. can you tell me waht could be the reason , is it due to any contamination or something like that....what should i do now.. kindly help me out..waiting for ur reply

Question: what are your primers specific to? I am assuming the gene from the start until its end, nothing more... Are you adding through tags any other sequences?
If I were you I would ignore it (it is not a big huge difference, it might be an artifact of the agarose; even though 600 bp it is a bit of too much of an artifact) and clone it and send it for sequencing to check what it is there. Or just send the PCR product for sequencing with the primers used for the PCR. Without seeing the primers and the plasmid sequence used for template, I cannot tell you more.

Andreea

#19 ascacioc

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Posted 30 August 2012 - 02:45 AM

Speaking about agarose artifacts aka DNA not running at the expected size:

http://www.protocol-...-than-expected/
http://www.protocol-...wer-than-uncut/




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