Hi,

From gel picture, it seems only some DNA was digested. Maybe need to check enzyme activity, just use plasmid with BamHI cut site, and test HamHI-HF is OK. If enzyme is fine, maybe increase enzyme volume, but remember total enzyme volume can not over 1/10 of total volume, and need to check star activity. 

Good Luck.

HI...

I think you should decrease the amount of DNA you are taking. make it 1 ug and 1 ul of enzyme. I usually work with 500 ng as well never had a problem. 

Your DNA has good quality and you should decrease the amount of your DNA or increase enzyme unit or increase restriction time. It is dependent to companies that you bought restriction enzyme. I used vivantise restriction enzyme. I used 1 unit of RE with a buffer that was 100% efficient and didn't have star activity and 1-2 ug of DNA and incubate them for overnight .

I hope be useful.

with genomic DNA I use:

Enzyme (RE)3ul

buffer 5ul

DW 6ul

DNA 6-10 ul (3000-5000ng/ul)=3-5ug

with plasmid, I use 1ul (RE) 

Good luck

my RE is the product of NEB R3136T, bam H1-HF along with cutsmart buffer. I have incubated at 37°C overnight. The concentration of my DNA is 400-1000 ng/ul testing by nanodrop.

Most people think that I should add more enzyme. but the enzyme is 100,000 unit/ml. The protocal said it is enough for 1 ug dna to be digested by 1 u enzyme. If I want to add more enzyme, the reaction volume should be larger, is it?

hi

you have to reconsider your digesting conditions.

when you digest 1microgram of genomic DNA you have to add 10 times more enzyme

so when you are digesting 8micrograms with only this low units of enzyme its normal digestion wont be effeicient and complete.

also you have to incease the volume

goodluck

What do you mean by adding 10 times more enzyme? You mean the valume or unit? I think it is enough for 1 ug DNA digested  by 10 uint enzyme, is it?

Hi, did you measure OD 260/280? What is the score? Is it possible that some proteins bind on DNA, so DNA could not digest by BamHI.

Today I tried  southern blot  again. 2 samples in 5 worked well. This time more enzyme was added(50unit enzyme/1 ug Dna). Some still has question. I think it is the Dna concentration problem.

for souther blot try the concentration that I mentioned up.

i tried my best to get enough DNA but 2000 ng

/ul is my maximum . What method do you use to exteact the dna?

1. is there any contamination in your DNA sample? Phenol or ethylene may affect the activity of enzyme

2.decease the amount of DNA, use 1-2 ug DNA per reaction, and 3 unit/ug enzyme

3. use a large volume, 50ul

4. use another enzyme as a control, like HindIII, and load a DNA marker

Other enzyme like Ecor1 can cut the dna.  Will 1-2 ug DNA per reaction work for southern blot. The minimum is 8 ug DNA per reaction recommanded by the protocal.

I mean you can use 1-2ug DNA per reaction to find out what the problem is, and fix it. If EcoRI works fine, it means your DNA sample is OK. I think you can use a new BamHI enzyme to repeat your experiment, and remember to use a large reaction volume. Normally, the final DNA concentration of digestion reaction is 100ng/ul.

thank you. I will try again.

Hi xi Song,

Generally for southern you need 3-4 ug DNA. but you can only load 20-30 ul volume in gel. that is what you are sticking with 30 ul restriction digestion volume!.

My suggestion is- increase the reaction volume from 30 ul to 90 or 100 ul. Then go for ethanol precipitation and dissolve your restricted digested DNA in small volume (eg-20 ul). then go for gel loading! 

The reason to increase the volume is that - out of 30 uL total volume you are using for res.digestion, you are adding 10-15 uL DNA (which contains 8 ug DNA). This 10-15 ul volume is usually TE buffer. However, in 30 uL reaction volume you have to add 3 ul of 10X cut smart buffer. So, your TE composition and its pH (10-15 ul DNA) disturbs the cut smart buffer environment. so without optimal buffer condition, your enzyme will not be fully active!

Hope this will help you!

Ramanan Devaraj