Restriction mapping - (Oct/13/2010 )

Can someone please explain how to do this i've got a basic idea but im just getting lost with all the numbers and lines im drawing

EcoRI 6kb BamHI = 6kb PstI = 6kb SstI 0.8,1.9,3.3
EcoRI + BamHI = 1.9,4.1
EcoRI + pstI = 2.6,3.4
BamHI + pstI = 1.5,4.5
sstI + EcoRI = 0.8,1.4,1.9,1.9
SstI + BamHI = 0.3,0.5,1.9,3.3
SstI + PstI = 0.7,0.8, 1.2, 3.3

the plasmid is supopsed to be 6kb in length..

thanks for any help

-ohsnap-

ohsnap on Wed Oct 13 09:10:36 2010 said:

Can someone please explain how to do this i've got a basic idea but im just getting lost with all the numbers and lines im drawing

EcoRI 6kb BamHI = 6kb PstI = 6kb SstI 0.8,1.9,3.3
EcoRI + BamHI = 1.9,4.1
EcoRI + pstI = 2.6,3.4
BamHI + pstI = 1.5,4.5
sstI + EcoRI = 0.8,1.4,1.9,1.9
SstI + BamHI = 0.3,0.5,1.9,3.3
SstI + PstI = 0.7,0.8, 1.2, 3.3

the plasmid is supopsed to be 6kb in length..

thanks for any help

For the sake of simplicity we'll start with EcoRI and as it only cuts once we'll say that it cuts at position 0kb.

Eco + Bam obviously only cuts twice so if Eco cuts at 0 then Bam must cut 1.9kb away (at position 1.9kb)

PstI cuts 1.5kb away from BamHI: this can either be at point 0.4kb (BamHI's1.9kb minus 1.5kb) or at 3.4kb (1.9+1.5).
---> But as PstI/EcoRIgives a 3.4kb frag this means PstI's site is at 3.4kb.

SstI/Eco gives a 0.8kb fragment but as this is missing for the Bam/Sst digest you can assume that BAm must cut in the middle of it, thus there are Sst sites 0.3 and 0.5kb away from the Bam site: either at 1.4/2.2 or 1.6/2.4. As the Eco/Sst digest gave a 1.4kb fragment we can assume it is the former option. (IE SstI cuts at 1.4kb and 2.2kb.)

In some ways we can ignore the last two digests that we have data for as the other two fragments from the Eco/Sst digest are equal in size so must be (negative) 1.9 and 3.8kb away from EcoRI. This gives us cut site of 4.1kb and 2.2kb (The second one we already know!)

In summary:-

EcoRI: 0kb
BamHI: 1.9kb
PstI: 3.4kb
SstI: 1.4, 2.2, 3.4kb

Hope this helps!

-Off the Leish-

Hi the teacher said we need to use bam at the first position so is it ok if i swap that with EcoRI if so what is the answer then

thanks so much for your help

-ohsnap-

There's this skill called problem solving, I suggest you try it.

-Off the Leish-