NEBCUTTER and expected size of fragments

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NEBCUTTER and expected size of fragments

Postby soulcalibur05 » Jan 24 2012 6:54 pm

how do i calculate expected size of fragments. , and the results for ddeI as below

DdeI sites in noname

# MS Cut position Site with flanks
---- -- ---------------- ----------------------------------------
1 12/15 2 catccaacat C^TCA_G catgatgaaa
2 35/38 25 aagctcctca C^TGA_G gactctgttt
3 329/332 319 atgtattacc C^TGA_G gacaaatatc
4 347/350 337 aaatatcatt C^TGA_G gggctgcatg
5 377/380 367 aaatatcaat C^TGA_G gggcccatcc

how they calculate? total bases is 385 bp
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Re: NEBCUTTER and expected size of fragments

Postby mchlbrmn » Jan 24 2012 11:20 pm

I don't really know what you are asking.
The 12/15 number means the first cut is in the top strand after base 12, and the bottom strand is cut after base number 15. Does that answer the question?
I'm sure you can find a more convenient format for the results from this or a different program.
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Re: NEBCUTTER and expected size of fragments

Postby relaxin » Jan 25 2012 9:19 am

I really do not understand the meaning of these numbers. Is the DNA linear or circular? To calculate the fragment size, just calculate the number of bp between the two DdeI sites.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
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Re: NEBCUTTER and expected size of fragments

Postby CrowSan » Jan 25 2012 11:20 am

Is the DNA linear or circular?
What is its (uncut) size?
It looks to cut 5x. In the 5' strand (from the limited data you have given the frag sizes would be:
23 bp (cuts at 12 and 35 bp = 35-12)
294 (cuts at 35 and 329 bp = 329-35)
18 bp (cuts at 329 and 347bp=347-329)
and 30bp (cuts at 347 and 377bp= 377-347)
There will also be one ore more fragments depending on whether this is a linear or circular DNA.
(i.e. if linear the "ends are cut off" - one will be 12 bp = the distance from the start of the 5 strand to the first cut site and the second will be the distance from the 377 site to the 3' end of the DNA)
If circular plasmid there will be one fragment of size = (total plasmid size minus all the fragment sizes removed)
(Not forgetting the 12bp still attatched to the DNA before the first cut site).
I suggest you draw it out on paper, mark on the 5' cut sites (first number in the ddle cut info i.e. 12, 35, 329, 347, 377)
and calculate the DNA pieces cut out.
I am guessing that what has condused you is the two "site numbers" for each cut site: as mchlbrmn has mentioned these refer to the site of cutting on the 5' and 3' strands (you only need to consider 5' or first number to calculate the fragment sizes).
What I have written may seem needlessly complicated but we lack sufficient info to answer you question clearly (hence all of the caveats above).
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Re: NEBCUTTER and expected size of fragments

Postby GelBandIt » Mar 03 2012 3:41 pm

If you use the "Custom Digest" function and select which enzymes you will cut with, on the results page, just click on "Fragments" under "List" in the bottom-right of the screen.

For this to work properly, you must specify whether the sequence is linear or circular (On the first sequence-entry screen)
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