Double digestion - (Jun/29/2007 )
Hi
Im trying to do double digestion for my Vector and DNA fragment with HindIII and EcoRI( both should work well with Buffer2).My vector size is 2.7 Kbp and my DNA fragment is 500bp.Unfortunately after doing PCR , I noticed that I have another restriction site for HindIII just after few base pair in front of my forward primer on my forward primer( that is restriction site for HindIII again)
I incubate 3 hours for the first enzyme and then add the second one for more 3 hours. Unfortunately, I haven't been sucessfull to do that.
my questions:
1- the reason/s
2- Does it make any differences to add which one first in this case?
2- After doing digestion how can I be sure it has been done( I should see one band for vector and DNA fragment as well??)
3-I have to do Ultra clean for both after double digestion?
It doesnt mind which enzyme you use first to digest the DNA with.
You could do a double digest it shouldnt matter except you have to take care of the glycerol content. too much could cause star activity for EcoRI.
I would run the digesting products on gel to clean it up.
How much vector and insert do you use for ligation? And what ratios are you using.
You could do a double digest it shouldnt matter except you have to take care of the glycerol content. too much could cause star activity for EcoRI.
I would run the digesting products on gel to clean it up.
How much vector and insert do you use for ligation? And what ratios are you using.
The first enzyme is EcoRI and the second one is HindIII. Actuall my main problem in cloning seems to be double digestion because I found out that my double digestion doesnt work well. because I have my vector uncut even after doing double digestion.
I didnt add glycerol to my double digestion . I just have : 1micro EcoRI+1 icro RNase A+ 8 mcro ddH2O+ 7 micro Vector+ 2 micro Buffer2 after incubation for 3 hours in 37 I add 1 micro HindIII and incubate for another 3 hours.
Because I should have just one band after double digestion for my vector( puc18) but I have 2-4 bands
I take it the buffer 2 you said are NEB buffer 2? Are your sites separated or overlapped? enough bp in between for the enzyme to cut? How much DNA is there in your 7ul vector? Since you don't really need much vector in your ligation, 2-4ug is mor than enough. Have too many bands likely is due to star activity. Make sure that total amount of glycerol (in your enzymes) will be at most 5%. I would recommend less enzymes, as little as you could get away with.
You are doing a 10ul digestion, but you got 2ul of 10xbuffer???? And also got total 3ul of enzymes???? If I am not wrong, enzymes are kept in 50% glycerol. Counting also pipetting error, star activity is a real risk.
Furthermore, as according to NEB information, as well as what we always do in our lab, when cut with EcoRI and HindIII, we use EcoRI buffer, not buffer 2 (which is for HindIII). I have never used RNAse and have never got any problem with double digestion. Add both enzymes together is fine (no need for sequential digestion), unless the cutting sites overlap.
You are doing a 10ul digestion, but you got 2ul of 10xbuffer???? And also got total 3ul of enzymes???? If I am not wrong, enzymes are kept in 50% glycerol. Counting also pipetting error, star activity is a real risk.
Furthermore, as according to NEB information, as well as what we always do in our lab, when cut with EcoRI and HindIII, we use EcoRI buffer, not buffer 2 (which is for HindIII). I have never used RNAse and have never got any problem with double digestion. Add both enzymes together is fine (no need for sequential digestion), unless the cutting sites overlap.
Hi Almasty
Thanks for your tips
Yes I used NWBUFFER 2. If you mean the cutting sites of the HindIII I mention those are too close ( just few bps apart) but cutting sites of the Hind III and EcoRI are apart.Acyually I've never measured my vectors DNA but I think its around 50 ng. I never add glycerol to my enzyme.
I did 20micro digestion overall and I add 8micro of 10X NEBUFFER 2.
I added 1 RNase+1 EcoRI+1 HindIII
I never add glycerol to my digestion. where it should be added?? is it in my buffer?? I dont think so.
If I want to use NEBUFFER EcoRI whats your recipe for double digestion( as you said I shouldnt add RNase)??I mean what amount of plasmid, enzymes and ddH2O you add? and in what order( enzymes and incubation time)? how about the incubation time??
PU18, I would suggest that you find out how much vector DNA you have there. Most preps should give you 100s of nanograms if not micrograms worth of DNA. So 50ng feels strange.
But in anycase, if the DNA concentration in the digest is too high, the DNA won’t cut. I would suggest that you increase your digestion volume to at least 50ul.
- Check DNA concentration and use enough DNA in your reaction. Don't use too much. It is a waste and the reaction won't be good.
- 20ul reaction and use 8ul 10x buffer? That is way too much. It is 10x buffer, so you need to dilute 10 times, right --> final concentration for your digestion is 1x buffer. In your case, 20ul reaction meant 18ul of DNA + enzymes + H2O and 2ul of 10x buffer, isn't it? Why did you use so much buffer? Too high concentration of salt is not good.
- You don't have to add glycerol into your reaction. Enzymes are supplied with glycerol, else they would have been frozen solid at -20oC (check NEB catalogue. NEB catalogue is VERY GOOD for cloning. HIGHLY RECOMMENDED if you are new at this. They give you a lot of tips, which buffer to use for double digestion, what need to be taken care of...). As I said, if I am not wrong, the enzymes will come in 50% glycerol. Calculating the amount of enzymes to add in your reaction so that the final concentration of glycerol has to be less than 5%, remember that you need to account for the possibility of pipetting error, too. Again, too much enzymes is NOT good either. Check the enzyme unit to see how much is enough for a given amount of DNA in a given time. Again NEB catalogue helps lots.
- Normally, I would just add about 3ug of DNA, 0.5ul of each enzymes, 5ul of 10x buffer, top up with Hs0 to 50ul, 37oC o/n, since I am lazy. Understand that those are fixed numbers. This much DNA means that I can do lots of ligation later, so after gel purification I will just frezze it down for future use.
You could do a double digest it shouldnt matter except you have to take care of the glycerol content. too much could cause star activity for EcoRI.
I would run the digesting products on gel to clean it up.
How much vector and insert do you use for ligation? And what ratios are you using.
The first enzyme is EcoRI and the second one is HindIII. Actuall my main problem in cloning seems to be double digestion because I found out that my double digestion doesnt work well. because I have my vector uncut even after doing double digestion.
I didnt add glycerol to my double digestion . I just have : 1micro EcoRI+1 icro RNase A+ 8 mcro ddH2O+ 7 micro Vector+ 2 micro Buffer2 after incubation for 3 hours in 37 I add 1 micro HindIII and incubate for another 3 hours.
Because I should have just one band after double digestion for my vector( puc18) but I have 2-4 bands
You are adding 1ul of enzyme and then adding an additional 1ul of enzyme which means you have around 5% or more of glycerol (from the enzymes). This could cause EcoRI star activity (reason for 2-4 bands). Try the same digestion in 50ul with the same amount of enzymes and have 5ul of buffer. This should solve the problem.
Hi everybody
Thanks for your tips. I reviewed the NEBUFFER and found out that I used NEBUFFER 2 for both ( although both enzymes have 100% activity)but according to neb.com :
Double Digest Recommendation(s) for EcoRI + HindIII:
Digest in NEBuffer EcoRI at 37°C.
But I dont have this buffer so is it possible to use lower concentration salt as first buffer +enzyme and then add the higher salt concentration buffer+enzyme???
I dont need to do ultraclean in between??
Thanks for your tips. I reviewed the NEBUFFER and found out that I used NEBUFFER 2 for both ( although both enzymes have 100% activity)but according to neb.com :
Double Digest Recommendation(s) for EcoRI + HindIII:
Digest in NEBuffer EcoRI at 37°C.
But I dont have this buffer so is it possible to use lower concentration salt as first buffer +enzyme and then add the higher salt concentration buffer+enzyme???
I dont need to do ultraclean in between??
I precipitate the DNA in between the digestion.