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Sequential restriction digestion of plasmid - (Feb/21/2009 )

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T C on Feb 23 2009, 02:06 PM said:

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

-jiajia1987-

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.

-shimshady-

jiajia1987 on Feb 22 2009, 09:57 PM said:

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.


shimshady on Feb 23 2009, 01:06 PM said:

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.

-perneseblue-

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

shimshady on Feb 24 2009, 02:36 AM said:

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.

-T C-

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

-friendly-

Hey

Maybe the DNA you are taking for setting up digestions is low in concentration, digest more of it.

Best
TC




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

-T C-

perneseblue on Feb 24 2009, 08:44 AM said:

jiajia1987 on Feb 22 2009, 09:57 PM said:

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.


shimshady on Feb 23 2009, 01:06 PM said:

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.


Hi there,

Thanks for your advice. I will take note of it. I have realized that the enzyme is really viscous and I always have a hard time trying to mix it in the reaction. How do you mix it? I usually swirl and pipette up and down and close the tube, tap on the base of the tube and spin down before I incubate it.

-jiajia1987-

T C on Feb 24 2009, 03:39 PM said:

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

shimshady on Feb 24 2009, 02:36 AM said:

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.



This is what I do at times and I realized that I can get really high concentrations by this method! Sometimes, after I have gotten the end product, I will suck it up and place it on the exact same spin column and let it stand for a min before spinning it again. This is to recover more DNA.

-jiajia1987-

jiajia1987 on Feb 24 2009, 05:26 PM said:

Thanks for your advice. I will take note of it. I have realized that the enzyme is really viscous and I always have a hard time trying to mix it in the reaction. How do you mix it? I usually swirl and pipette up and down and close the tube, tap on the base of the tube and spin down before I incubate it.


About the same. Although, once it is in the tube, I vortex the solution (assuming that the plasmid and not a large BAC)

-perneseblue-

perneseblue on Feb 25 2009, 09:34 AM said:

jiajia1987 on Feb 24 2009, 05:26 PM said:

Thanks for your advice. I will take note of it. I have realized that the enzyme is really viscous and I always have a hard time trying to mix it in the reaction. How do you mix it? I usually swirl and pipette up and down and close the tube, tap on the base of the tube and spin down before I incubate it.


About the same. Although, once it is in the tube, I vortex the solution (assuming that the plasmid and not a large BAC)


cool. I didnt know that I could vortex the solution. I was always scared that the DNA would get sheared.

-jiajia1987-
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