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Common RE site on complementary strands in a Vector - (Nov/10/2013 )

Hi Experts

 

I want to digest pCAMBIA1301 with EcoR1. One RE site for this enzyme is present on coding strand and one at a distant position on the non-coding strand. Will it cut the vector at both positions after overnight digestion reaction?

Please reply

-awan-

Hi Experts

 

I want to digest pCAMBIA1301 with EcoR1. One RE site for this enzyme is present on coding strand and one at a distant position on the non-coding strand. Will it cut the vector at both positions after overnight digestion reaction?

Please reply

I have serious doubts whether you understand how a RE works!

 

 

it will cut all the sites it recognises...

 

I am not sure why you speak of the coding strand and the non coding strand....

 

How do you think it works?

-pito-

pCAMBIA1301 has an AflII site on one strand and an EcoR1 site on the other strand. The sequence of these two REs is complementary to each other. so, my question is that weather AflII will cut the vector at both these sites. 5'-CTTAAG-3' for AflII and 5'-GAATTC-3' for EcoR1.  

-awan-

pCAMBIA1301 has an AflII site on one strand and an EcoR1 site on the other strand. The sequence of these two REs is complementary to each other. so, my question is that weather AflII will cut the vector at both these sites. 5'-CTTAAG-3' for AflII and 5'-GAATTC-3' for EcoR1.  

Look here: http://en.wikipedia.org/wiki/EcoRI

 

and look at this: http://upload.wikimedia.org/wikipedia/commons/7/78/EcoRI_restriction_enzyme_recognition_site.svg

 

are you starting to understand what I mean?

 

I think you are bit confused!

 

It if cuts "1" strand , it will also cut the other strand...

 

THe site of recognition is not just found on one strand, its on both strands.

I find it strange that you say that the RE recognition site is on 1 strand and not on the other...

 

5'-GAATTC-3' for EcoR1.  , this means that EcoR1 will cut when it "finds" a GAATTC sequence....

1 strand: 5 => 3 or second strand: 3 =>5 (or from the other side, 5 =>3 ...) An enzyme does not "know" what "strand" it is....

 

5'-CTTAAG-3' for AflII , so AflII will simple recognise CTTAAG in the 5 => 3 direction or GAATTC in the 3 => 5 direction..

 

 

I am not sure you really understand how they cut.

They cut both strands, not just 1 strand....

 

Check this too: http://en.wikipedia.org/wiki/Restriction_enzyme

 

I think you are bit confused about how they cut.

-pito-

Dear pito

 

Thank u very much for your guidance

 

Yes I am quite confused

actually I checked the sequence of pCAMBIA1301 and cloned my insert of 1.5 kb at the Nco1 and AflII. later I found that the EcoR1 site, located at a distant site in the same vector cuts the same sequence.My quetion is that weather AflII will cut at two site and give a fragment (2 bands) or just give a linearized vector (single band). I think It should give 2 bands, but I got a single band. please if you can clear my understanding.  

-awan-

Dear pito

 

Thank u very much for your guidance

 

Yes I am quite confused

actually I checked the sequence of pCAMBIA1301 and cloned my insert of 1.5 kb at the Nco1 and AflII. later I found that the EcoR1 site, located at a distant site in the same vector cuts the same sequence.My quetion is that weather AflII will cut at two site and give a fragment (2 bands) or just give a linearized vector (single band). I think It should give 2 bands, but I got a single band. please if you can clear my understanding.  

 

 

How did you do it?

 

Did you use both RE to digest the vector?

I dont know the sequence, however:

I checked the vector map here: http://www.cpgbiotech.com/res/cpgbio/teres/201107/20110731151458418.jpg

 

I dont see what you mean with 2 EcoRI restriction sites...

I only see one in the MCS.

 

Same for Nco 1 , I only see 1 cut place...

 

AflII, I dont see this at all (but maybe a similar place is cut by another RE).

 

 

Anyway: if you would use the plasmid that I show here and you would cut with EcoRI and Nco 1 you would end up with 2 pieces: you cut at both RE sites and thus you would have a "long piece" of plasmid from Nco1 to EcoRI and 1 "short piece" from EcoRI to Nco1

(seen from right to left , so from Nco 1 to Ecori)

 

So what is happening (I assume this is the goal, but not sure since I dont know the protocol) is that you cut the plasmid and "remove" the "short piece" and put the insert in stead of this short piece.

So you end up with the insert in between the EcoRI and Nco1 (where the short piece used to be).

 

I hope this makes sense?

 

 

 

 

I looked the RE up and found this:

 

aflII

5' CTTAAG
3' GAATTC

 

Cuts like this:

 

5' ---C   TTAAG--- 3'
3' ---GAATT   C--- 5'

 

EcoRI

5' GAATTC
3' CTTAAG

 

Cuts like this:

5' ---G   AATTC--- 3'
3' ---CTTAA   G--- 5' 

 

turned around it would be (simple put the 3'-5' on top and "flip" it)

 

5' ---G  AATTC--- 3'
3' ---CTTAA  G--- 5' 

 

 

==> see ?

The sequences are the same, but they do not cut the same way!

 

 

ncoI

5' CCATGG
3' GGTACC

cuts like this:
5' ---C   CATGG--- 3'
3' ---GGTAC   C--- 5'

 

 

 

THey do not cut the same way....

 

 

So even if they recognise the same nucleotide sequence, it does not mean they cut the same way....

-pito-

AflII and EcoRI cut very different sequences. You need to review basic structure of DNA, and in particular the concept of reverse complements. EcoRI cuts at the sequence GAATTC. When you reverse complement that sequence, you get GAATTC (it is a "palindromic" sequence). Restriction enzymes bind to that specific sequence. They do not know or care if the sequence is on one strand or the other -- in this case, it binds on both strands (at the same sequence!! but in opposite orientation).

 

AflII cuts at the CTTAAG sequence. Note that when you reverse complement this sequence, you get CTTAAG. This sequence is COMPLETELY DIFFERENT and UNRELATED to the sequence GAATTC.

-phage434-

       
        Hi Pito and phage434
 
Thank you so much. Finally I got it and Its all because of your guidance and teaching. Today I rewrite the restriction sequences and while writing I understand where I was confused. Thanks again for the help.
 
 
 

-awan-