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DNA loss in digestion - I lose lot of DNA in digestion (Aug/06/2007 )

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hello every body ..
I have a problem..
I have PCR product..I run 1uL of gel purified PCR product again on gel to check the concentration of purified pruduct..I ca nsee very dark bands.(150ng/uL)
Then I use this dna for double enzyme digestion. 15uL/60uL reaction....The enzyme cuts 8bps frm both ends of the fragment..there is no internal site for the enzyme in the fragment..
but after digestion when I run all the 60uL reaction volume on gel I get very weak bands..and after gel purification the product is is very low..
I also see some dark bands above the wells on the gel..showing like some of the the dna is moving in reverse.....

i need your help..somebody please explain it ..i need urgent help...

pleassseeeeeeee

thank u in advance

ALi

-alichem-

Can you confirm the PCR DNA conc. is 150ng/ul? If you used 15ul of the DNA, it would mean that you have used more than 2ug of DNA.

Unless there is some contamination which could have degraded your DNA, I would verify the initial DNA concentration.

If you start with a low amount of DNA, then you will get even lower amounts of DNA by the time its digested and purified on gel.

Good Luck !!!

-scolix-

I determined the DNA concentration so many times from Gel bands..and every time the concentration was above 100ng/uL.
after gel purification of PCR products I do the following step to determine the DNA concentration....

1. 0.5uL DNA
2. 4.5uL H2O
3. 1uL 6X gel loading buffer.

I load this on gel with 1 KB ladder 1uL..and I get very nice bands for the DNA ..
then from this DNA i use 15uL for GEl Digestion...
I expect a very dark band after gel digestion..but i get very weak band on gel and after gel purification the concentration is further lowered ..

-alichem-

do you directly run your digest onto the gel for gel purificatioin? Or do you percipitate the DNA first? Is there a possibility of losing the DNA?

Could this be a case of insufficient staining of the gel during the gel purification? Do you run a ladder on the purification gel? How do you excise your DNA band? Open UV sourse right?

-perneseblue-

I think you could digest your DNA in a smaller volume (not 60┬Ál). and a good trick is to precipitate your DNA (e.g. after gelextraction) with glycogen (carrier) and resolve it in smaller volumes.

-moljul-

QUOTE (perneseblue @ Aug 6 2007, 11:38 PM)
do you directly run your digest onto the gel for gel purificatioin? Or do you percipitate the DNA first? Is there a possibility of losing the DNA?

Could this be a case of insufficient staining of the gel during the gel purification? Do you run a ladder on the purification gel? How do you excise your DNA band? Open UV sourse right?


I heat the digested product at 70C for 10 minutes and then mix the gel loading buffer contaning ETDA. and load the digestion product on gel using 1Kb Ladder.....
i do not precipitate the DNA ...is it required before loading it on a gel ?

-alichem-

no, not really. Percipitating your DNA first gives you nicer bands. However it is possible to be careless at this point and lose the pellet or to not full redisolve the pellet, causing the DNA not to run into the gel.

How does the band strength compare to the ladder on the purification gel? Are you staining long enough?
Is the gel clean? Is the l;oading buffer clean? Might it be possible that your DNA is being degraded? Finally are you certain you really do have lots of DNA to begin with? By setting long exposure times on the camera of the gel documentation system, one can make even faint bands appear strong.

Something is amist here, DNA can not simply vanish. It has to be going somewhere

-perneseblue-

I agree with perneseblue,

You are losing DNA somewhere or your input is low to begin with. Try to figure it out. DNA precipitation is not going to solve the problem. And you will still lose a small percentage of DNA after precipitation.

-scolix-

QUOTE (scolix @ Aug 8 2007, 12:27 PM)
I agree with perneseblue,

You are losing DNA somewhere or your input is low to begin with. Try to figure it out. DNA precipitation is not going to solve the problem. And you will still lose a small percentage of DNA after precipitation.


hello all..
Let me explain my problem again....
1. I run a PCR product on a gel and it gives a strong band as compared to DNA ladder...Exposure 8s for all gels usinf biorad gel imager..for cutting bands I use transilluminator..it take almost 5s to cut band on UV transilluminator...
2. I gel Purify the bands using zymoclean kits..
3. After gel purification I use 1uL on gel to determin concentration using Gelloading dye...8s exposure to UV ...comared to DNA ladder the band is tronger than DNA ladder when I load 1 uL dna. (I determine the concentration from this band so therefore DNA Loss is not a problem as I determine concentration after gel purification..so this concentration is the recovered DNA conc.)

4. Then I 15 uL of this gel purified DNA for Double enzyme digestion.(clearly 15uL DNA should give much tronger band as 1uL dna gives band stronger than DNA ladder band)
5. After digestion with bamHI and HindIII..I heat the digestion mixture at 70C on PCR machine for 10minutes..
6. Then I load thereaction product on a gel. the reaction product contains 15uL..even if I lose 50% during digestion still 7uL DNA should be there which again should give much stronger band as compared to 1uL DNA which I loaded before digestion..
7. The Enzyme cuts 8bps from both end of the DNA ..DNA size is 2Kb..


8. I have run so many gels I never see any bands above the wells on a gel ..but when I lead a digestion prouct on gel i see bands on a gel above the wells also...(thats showing some is moving in opposite dorection)


hope this is enough to explain my problem..
thank you sooooooooooo much for your help
I really love this forum and all the sincere people
wish u all the best

Ali

-alichem-

Any hint of a smear in the gel? Could you try to change the elution buffer when you purify from the gel. It may contain some contamination which might work efficiently in the presence of the enzyme buffers and result in loss of DNA. its just a guess.

Good Luck !!!

-scolix-

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