failed restriction digests - (Sep/29/2008 )

i've been working with some DNA extractions and trying to analyze them with restriction digests to see if the DNA is clean. the DNA has been extracted from chloroplasts. i have very little DNA to begin with so perhaps that's the problem.

the first time i tried it, i was working with 1.7ng/uL DNA. i was able to see a band before digestion. after digestion there were no bands at all. I mixed together

76 uL DNA
4 uL 10x restriction buffer
3 uL HindIII (10 units/uL)

but i got no bands. I stopped the reaction with 5 uL of the buffer after incubating for 2 hours at 37 C.

So, I ran another extraction and this time got 13 ng/uL DNA. I thought definitely i should get something. Wrong! This time i used:

8.5 uL DNA
1.0 uL 10X restriction buffer
0.5 uL HindIII (10 units/uL)

and I incubated for 2 hours at 37 C but stopped the reaction with 15 minutes at 75 C.

In both cases I don't see any bands whatsoever. Is it possible that it is digesting but that I'm starting with so little DNA that I cannot see the bands? or are my recipes wrong?

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let's look at your 2nd case more. did you run a gel before the HindIII digestion for the 2nd case?
13ug/ul is WAY too high to me . 13000ng/ul ?!
you might be getting RNA who knows instead of DNA @! check A260/280 ratio?!

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oops! i actually meant to say ng/uL not ug/uL. i went back and edited it.

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let's look at your 2nd case more. did you run a gel before the HindIII digestion for the 2nd case?
13ug/ul is WAY too high to me . 13000ng/ul ?!
you might be getting RNA who knows instead of DNA @! check A260/280 ratio?!
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i didn't run a gel the second time because i was so sure that I had DNA because the extracted chloroplasts were very abundant and clean. my ratio was also 1.76 which i thought was good enough for my purposes. i just wanted to practice restriction digests and get a feel for the procedure.

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so this dna is not pcr nor plasmid /// but more like genomic. did u know wat happen when u cut genomic w/ an enzyme? u get a smear//

u mihgt have something but u can't see it.

send a biodocit image over once u did ur 2nd round digestion. make a lane ofr " undigestesd" .

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It is possible that the amounts of DNA being extracted is too low that you are unable to see it.

If you measured 0.0017ng/ul, I don't think its accurate. Its too low to be measured.

If you did measure 13ng/ul, then use atleast 10ul and run it on get to see DNA bands, If you dont see anything, then the DNA amounts r too low.

Better do restriction digest in atleast 20ul reactions. And your first digest has the wrong amount of buffer( I guess it might be typo).( for a 80ul reaction, you need 8ul of a 10x buffer and the total reaction should be 80ul which means including your enzyme, the reaction volume is 80ul)

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The digestion willn't tell you if DNA is clean or not. For all nucleic acid extraction is the ratio when reading in the spectrophotometer that will tell that or if get lucky and your lab have money a affimetrix analyser will also help or for RNA a gel will help. But the better and cheaper is the lectures of the spectrophotometer.

For chloroplast DNA you should have a higher concentration and yield. Check the extraction protocol carefully to check if you are making a mistake in one of the step. I'm not an expert in vegetable DNA extraction, but in here there is a lot of people that can help you. Make sure you have chloroplast DNA and not gDNA.

Back to the Restriction enzymes:

The recipes aren't at the right concentration, let the digestion incubate longer and make sure is at the right temp.
The buffer of the RE come in 10X concentration that means 1µL of buffer/10µL of reaction.
For what you will use the DNA???for PCR, cloning???Maybe with that we can help more.

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as i understand it, total or nuclear DNA will give you a smear but chloroplast or mitochondrial DNA will digest into many bands.

also, i ran a gel with the first sample which included undigested material and i was able to see a band. i loaded the wells with 5, 10 and 15 uL and i was able to see a band in 10 and 15 uL.

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you're right, it wasn't 0.0017ng, it was 1.7 ng. that was just my conversion to ug/uL. however, i was able to see a band for this when i ran an undigested sample. i loaded three wells with 5, 10 and 15 uL and was able to clearly see a faint band with 10 and 15 uL. because of this, i didn't bother to run a gel of the undigested second sample since i had 13 ng/uL.

for the first digest i was being cheap with the buffer. i found a recipe online that asked for 1-2 uL of buffer for a 20 uL sample. so i used the lower number so as not to dilute my DNA since i had so little.

on the topic of recipes, i have another question. if my enzyme is 10 units/uL, does that mean that i have to treat is as a 10X? that is, when i make a reaction mixture and i want calculate how much enzyme to add do i have to take into consideration the amount of DNA in my mix, the amount of buffer, or the total volume? It seems that there can't be a number that would satifsfy all three.

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Just add 1 ul. 1 unit means can digest 1ug linear DNA in X time at Y temp ( i forogt x n y ). for 20ul-50ul i like to add 1ul. it should be more than enough. no need to be cheap with all these things. normally i find it very hard to exhaust the restrictino enzyme in my lab . They normally will last way beyond their expiry date.

I really doubt chloroplast is not like genomic for this reason. If you get only one band and no smear or whatsoever, and by that i mean a very CLEAN band then it's weird lol. how smeary it gets depends on how big the genome size is and how often the RE cut it.

I remember cutting bacterial gDNA with EcoRI before , not much smear the band at the bottom still quite big . but when i combine EcoRI and AFl III. i got really nice smear which is good coz i wanna proceed with Southern Blot

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