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Sequential restriction digestion of plasmid


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#16 T C

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Posted 24 February 2009 - 01:13 AM

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

#17 jiajia1987

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Posted 24 February 2009 - 05:26 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.


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.

#18 jiajia1987

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Posted 24 February 2009 - 05:28 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.


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.

#19 perneseblue

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Posted 24 February 2009 - 05:34 PM

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)
May your PCR products be long, your protocols short and your boss on holiday

#20 jiajia1987

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Posted 24 February 2009 - 05:53 PM

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.

#21 friendly

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Posted 24 February 2009 - 10:44 PM

Thanks TC, can u show me what is the ratio u used for restriction digest?
Normally I will put

7ul of ddH2O
1ul 10x buffer
1ul sample
1ul of Enzym (EcoRI or BamHI)

my sample concentration were around 1.8-2.0 ug/ml

will it be too little??
Please let me know your sample preparation please.
and can u see clear fragmets when digested??

Thank you very much


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



#22 T C

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Posted 25 February 2009 - 12:07 AM

Hey

We do a lot of cloning in the lab so don't really quantitate the DNA before digestions. I normally grow a 3ml culture for 12 hrs and do a miniprep without using the column and resuspend in 30ul water.

Take 0.5 ul of this DNA, 2ul NEB buffer(1X), 0.3 ul enzyme, 0.2 ul BSA(if required)(1X) and rest is water in a 20ul digest. Digest 8-13 hrs for a prep digestion and put up multiple digestions if you want more of the digested product..

I see a very distinct band on the gel.

Let me know if you want to know anything else.

Best
TC

#23 perneseblue

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Posted 25 February 2009 - 01:52 AM

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

Lightly vortexed. Just touched the tube.
May your PCR products be long, your protocols short and your boss on holiday

#24 jiajia1987

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Posted 25 February 2009 - 02:28 AM

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

Lightly vortexed. Just touched the tube.


Yep! will do that in future! thanks heaps!

#25 Ecoli0157

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Posted 25 February 2009 - 12:07 PM

Just curious...
So after linearinzing the plasmid by enz digest, is it going to exist in super-coiled form or just circular DNA? Or The plasmid becomes super-coiled only after transformation because when I miniprep, it is in super-coiled form.

#26 friendly

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Posted 25 February 2009 - 10:49 PM

Thanks TC for ur reply, I normally use specific buffer for the enzyme ..My enzyme is from Promega, will it be less efficient??
If you don't mind can u send me ur gel photo which contain digested DNA??
THank you very much.

Hey

We do a lot of cloning in the lab so don't really quantitate the DNA before digestions. I normally grow a 3ml culture for 12 hrs and do a miniprep without using the column and resuspend in 30ul water.

Take 0.5 ul of this DNA, 2ul NEB buffer(1X), 0.3 ul enzyme, 0.2 ul BSA(if required)(1X) and rest is water in a 20ul digest. Digest 8-13 hrs for a prep digestion and put up multiple digestions if you want more of the digested product..

I see a very distinct band on the gel.

Let me know if you want to know anything else.

Best
TC



#27 T C

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Posted 26 February 2009 - 12:55 AM

Hey

I don't think the company makes a difference as long as you stick to teh protocols. Here is a gel pic of one of the clones digested with Nde I/ Hind III. But I wonder how this would help. B)
Lane 1,2,3: digestion in triplets
4: 1 kb ladder
5: 100 bp ladder

Best
TC

Attached Thumbnails

  • digested_clone.jpg


#28 friendly

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Posted 26 February 2009 - 06:58 PM

Thanks for your prompt reply.
It is helpful by looking at the photo because I know that your DNA size are less than 1Kb...
I have DNA that are very big..more than 1kb at least...
have u try digest DNA that have more thab 1kb ???
Would u still see the distinct band for DNA more than 1kb??

Thanks TC

Hey

I don't think the company makes a difference as long as you stick to teh protocols. Here is a gel pic of one of the clones digested with Nde I/ Hind III. But I wonder how this would help. :D
Lane 1,2,3: digestion in triplets
4: 1 kb ladder
5: 100 bp ladder

Best
TC



#29 T C

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Posted 26 February 2009 - 08:17 PM

Hey

My mistake....should have read the ladder for you

Read it this way

Lane 1,2,3: digestion in triplets
4: 1 kb ladder, from top read it this way: 10kb, 8kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1.5kb, 1kb, 500bp
5: 100 bp ladder, from top read it this way: 1.5kb, 1.2kb, 1kb, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, 200bp, 100bp.

So the bands are ~2.8 and ~5.5 kb (can dig for the exact sizes if u want).

Best
TC


[quote name='friendly' date='Feb 27 2009, 09:28 AM' post='17103']
Thanks for your prompt reply.
It is helpful by looking at the photo because I know that your DNA size are less than 1Kb...
I have DNA that are very big..more than 1kb at least...
have u try digest DNA that have more thab 1kb ???
Would u still see the distinct band for DNA more than 1kb??

Thanks TC

#30 friendly

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Posted 27 February 2009 - 12:58 AM

oh..
Thanks again.
TC, later I will attach my photo which I digested with EcoRI and please advice me
I am digesting Banana DNA..
Thanks




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