Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
* * * * * 1 votes

Sequential restriction digestion of plasmid


  • This topic is locked This topic is locked
58 replies to this topic

#1 shimshady

shimshady

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 37 posts
0
Neutral

Posted 21 February 2009 - 01:32 PM

I am trying to digest pET-15b vector using NdeI and BamHI. Sequential digestion is recommended for this so im going to do NdeI then BamHI after. My question is, is it necessary to purify the now linear plasmid after the first digestion or can i just go on and add the second restriction enzyme, having both in their the same time.

My second questions is how do the circular and linear DNA differ on an agarose gel. Thanks

#2 perneseblue

perneseblue

    Unlimited ligation works!

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 578 posts
17
Good

Posted 21 February 2009 - 01:58 PM

no, you do not need to purify the linear plasmid after the first digest. NdeI like NEB buffer4. And BamHI can work in buffer 4. Thus the sequential digest begins with NdeI. Once the DNA is linearised, add BamHI and continue the digestion.

An uncut preparation of plasmid DNA contains plasmids in several topologies. cut linear DNA, supercoiled circular DNA and nicked open circular DNA. Supercoiled DNA moves faster than linear DNA as it is more compact. Then comes cut linear DNA (due to shearing). And the slowest of all is the nicked open circular DNA, which has lost its twist and is now a floppy loop. A big circle has greater difficulties migrating through the agarose mesh.

Thus circular DNA doesn't run like linear DNA. And as the DNA ladders are linear DNA, you don't get the expected size.

If you are unfamiliar of how circular DNA looks, I suggest a sample of uncut DNA be run as control.

http://humgen.wustl....age/plsmid3.gif
May your PCR products be long, your protocols short and your boss on holiday

#3 scolix

scolix

    a student

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 314 posts
5
Neutral

Posted 21 February 2009 - 11:20 PM

I am trying to digest pET-15b vector using NdeI and BamHI. Sequential digestion is recommended for this so im going to do NdeI then BamHI after. My question is, is it necessary to purify the now linear plasmid after the first digestion or can i just go on and add the second restriction enzyme, having both in their the same time.


Inaddition to pernseblue's suggestion, remember not to exceed the glycerol percentage after adding the second enzyme.

#4 WHR

WHR

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 49 posts
0
Neutral

Posted 22 February 2009 - 01:09 AM

Hi,
It should be ok to perform double digestion at the same time in a single tube.
If the buffer used are not compatible, digest with the first RE with low salt buffer in a small volume, then the second RE with high salt buffer in a increased volume. No need of purification or extraction in between.

Edited by WHR, 22 February 2009 - 01:13 AM.


#5 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 22 February 2009 - 02:10 AM

Hi,

I am cutting my pET22b vector with Nde1 and BamH1 as well. What I would recommend is sequential digestion too. However, it would be advisable to do a PCR cleanup in between. BamH1 can work in Buffer 4 but its activity is best with Buffer 3. Plus, BamH1 has the problem of STAR activity and you need to remember to add BSA into the reaction to let BamH1 work.

I tried double digestion before, but it didn't work out as well. Hope this helps.

And, I am sorry for this additional question. But how long do you digest the plasmid? I do a 1.5hr digestion with Nde1 first and then a 1.5hr digestion with BamH1. Sometimes, I do a 2hr digestion during the sequential digestion.

The only problem I have is that a full digestion is not ensured and I do get self-religated vector at times. I am thinking of doing an overnight digestion, but this is not recommended for double digestion. Anybody has any ideas?

Edited by jiajia1987, 22 February 2009 - 02:12 AM.


#6 perneseblue

perneseblue

    Unlimited ligation works!

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 578 posts
17
Good

Posted 22 February 2009 - 02:31 AM

I do overnight digest (double digest or otherwise) just fine. I think it might be due to the amount of enzyme I am using (very little) in a large volume.

x volume DNA
y volume ds water
5ul of 100x BSA
1ul enzyme
10ul of 10x NEB bufer

total volume 100ul.
May your PCR products be long, your protocols short and your boss on holiday

#7 rkay447

rkay447

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 177 posts
20
Excellent

Posted 22 February 2009 - 05:00 AM

To prevent religated vector you should Cip or Sap treat the vector. After your digestions are done, just add one ul of Cip/Sap enzyme to the buffer already present. Incubate at 37 degree for one hour and continue with purification and ligation. This removes the 5' phosphate which is critical for ligation. Now, the vector can only ligate with the insert since the insert has a 5' phosphate after digestion.

#8 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 22 February 2009 - 09:57 PM

I do overnight digest (double digest or otherwise) just fine. I think it might be due to the amount of enzyme I am using (very little) in a large volume.

x volume DNA
y volume ds water
5ul of 100x BSA
1ul enzyme
10ul of 10x NEB bufer

total volume 100ul.


would it be possible for me to have a total volume of 50ul instead of 100ul? like 0.5ul BSA, 1ul Nde1, 1ul BamH1, 5ul NE Buffer 3, plus the DNA and water? I am asking this with regards to overnight digestion.

#9 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 22 February 2009 - 09:58 PM

To prevent religated vector you should Cip or Sap treat the vector. After your digestions are done, just add one ul of Cip/Sap enzyme to the buffer already present. Incubate at 37 degree for one hour and continue with purification and ligation. This removes the 5' phosphate which is critical for ligation. Now, the vector can only ligate with the insert since the insert has a 5' phosphate after digestion.


I thought of using Cip or Sap, but my supervisor does not provide me with these two. What is the difference between Cip or Sap? :D

#10 T C

T C

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 277 posts
0
Neutral

Posted 22 February 2009 - 10:06 PM

Hey

Calf alkaline phosphatase and shrimp alkaline phosphatase, SAP does work better but has problems with buffer excahnge and involves extra steps, CIP is easy to use as it works in all NEB buffers, so just add, incubate and run on gel.

When it come to BamHI, I always go for sequential digestions, unless its a check digestion.
TC

#11 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 22 February 2009 - 11:15 PM

Hey

Calf alkaline phosphatase and shrimp alkaline phosphatase, SAP does work better but has problems with buffer excahnge and involves extra steps, CIP is easy to use as it works in all NEB buffers, so just add, incubate and run on gel.

When it come to BamHI, I always go for sequential digestions, unless its a check digestion.
TC


Dear T C,

Thanks for your reply. :D

#12 shimshady

shimshady

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 37 posts
0
Neutral

Posted 23 February 2009 - 01:06 PM

I have another question when you gel purify the digested plasmid, do you load all your plasmid into one lane ore seperate lanes. I mean i know there are different volumes to which a lane can hold but do you try and do as much as possible or is better to do multiple? i have 60 ul reaction.

#13 perneseblue

perneseblue

    Unlimited ligation works!

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 578 posts
17
Good

Posted 23 February 2009 - 04:44 PM

would it be possible for me to have a total volume of 50ul instead of 100ul? like 0.5ul BSA, 1ul Nde1, 1ul BamH1, 5ul NE Buffer 3, plus the DNA and water? I am asking this with regards to overnight digestion.


It should work. I don't see a reason why not. However be really careful when adding the enzyme. Since it comes in 50% glycerol it is highly viscous. So there might be enzyme stuck on outside of the pipette and may result in one adding more (alot more) enzyme that desired. And lots of glycerol can inhibit enzymatic activity or cause star activity.


I have another question when you gel purify the digested plasmid, do you load all your plasmid into one lane ore seperate lanes. I mean i know there are different volumes to which a lane can hold but do you try and do as much as possible or is better to do multiple? i have 60 ul reaction.


I take some tape and tape together several teeth of a comb to produce one really really big well. When gel purifying, you don't want the band to be over loaded. If a band is overloaded, really dense DNA bands can drag fragment that are not the same size with it.
May your PCR products be long, your protocols short and your boss on holiday

#14 T C

T C

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 277 posts
0
Neutral

Posted 23 February 2009 - 11:39 PM

Hey
I load in separate lanes and dissolve them independently in QG buffer for qiagen, add isopropanol and now pool everythign on column. Add-Spin-Discard till all the vials are done.

TC

I have another question when you gel purify the digested plasmid, do you load all your plasmid into one lane ore seperate lanes. I mean i know there are different volumes to which a lane can hold but do you try and do as much as possible or is better to do multiple? i have 60 ul reaction.



#15 friendly

friendly

    member

  • Active Members
  • Pip
  • 16 posts
0
Neutral

Posted 24 February 2009 - 12:40 AM

Just curious question...
during all of your digestion...can you all see the abvious fragments on the gel ??
I couldn't view the clear fragments from the gel ...but using lamda DNA I can see the clear fragments.

I am using enzyme EcoRI and BamH1..

Thank you




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.