Digestion, clean up lead to fewer DNA! Help... - (Dec/15/2008 )
Dear all,
I am constructing vector pGADT7 with a 105bp insert. I have chosen EcoRI and BamHI sites for inserting the product (105bp) into the vector and these sites are present both in the vector and insert. I have got the insert cut out of PCR2.1 vector by double digestion. With the same insert I have constructed another vector previously. So there should'nt be any problem with insert. I did double digestion for this pGADT7 vector and did ligation with the same insert. After transforming (purified and unpurified ligation mix), it didnt gave any colony. But I was confidant of the digestion as i included the controls with a single enzyme for the digestion.
So I decided to do sequential digestion for my vectors. Breifly I took around 10 micrograms of dna (5 microgram in each tube) and set up a 100 miclitre reaction with EcoRI (Promega buffer H) first. incubated for 2 hours at 37 and loaded a aliqout on gel, confirmed complete digestion. then using qiagen pcr purification kit I purified this digest. eluted in 50 miclitres(again two slots). With this i went for BamHI digestion(Promega buffer E) and after 2 hours of incubation, electrophoresed in .8% LMagarose and did gel purification. Finally I obtained only about 450 micrograms of vector.
This vector I used for ligation again and waiting for the result.
Now my questions are:
1. Is there any way to improve the yeild of DNA in doing sequential digestion. Is there any alternate protocol?
2. Whether sequential digestion instead of double digestion will make any significant difference?
There are many experienced members in this group who could really help in such situations. Answering such questions may help starters lile me!
Thanks in advance!
Could you rephrase your question, i don't quite understand. Give enough time for the enzyme to digest all your DNA. Did you check your sequential digest to make sure that all the DNA was cut? The method you are using also you to check. Cut more DNA?
I think there is an error. Do you mean 450ng of vector? After running the gel to purify the DNA, do you get a nice strong band? Is the DNA "lost" at this step or at the step when you extract the DNA from the gel.
Are you using a DNA purification column to free your DNA from the gel matrix? Or are you using a crushed gel centrifugation method?
It depends on the enzyme pair used and the number of base pairs between the two restriction sites. Some restriction enzymes like NotI and NdeI require at least 8bp skirting the restriction site for the enzyme to efficiently cut the restriction site. So if you have one restriction site too close to another, it helps a lot if the enzyme that needs the most bp cuts first. And there is of course restriction enzyme pairs that work noncompatible restriction buffers or at different temperatures (eg BssHII which works at 55 Celsius and SmaI at 25 C).
Otherwise, there really isn't much difference between sequential digest and double digest. In your situation BamHI and EcoRI there is no reason to do a sequential digest. Those two enzymes are both very robust and work well together.
Could you rephrase your question, i don't quite understand. Give enough time for the enzyme to digest all your DNA. Did you check your sequential digest to make sure that all the DNA was cut? The method you are using also you to check. Cut more DNA?
I think there is an error. Do you mean 450ng of vector? After running the gel to purify the DNA, do you get a nice strong band? Is the DNA "lost" at this step or at the step when you extract the DNA from the gel.
Are you using a DNA purification column to free your DNA from the gel matrix? Or are you using a crushed gel centrifugation method?
It depends on the enzyme pair used and the number of base pairs between the two restriction sites. Some restriction enzymes like NotI and NdeI require at least 8bp skirting the restriction site for the enzyme to efficiently cut the restriction site. So if you have one restriction site too close to another, it helps a lot if the enzyme that needs the most bp cuts first. And there is of course restriction enzyme pairs that work noncompatible restriction buffers or at different temperatures (eg BssHII which works at 55 Celsius and SmaI at 25 C).
Otherwise, there really isn't much difference between sequential digest and double digest. In your situation BamHI and EcoRI there is no reason to do a sequential digest. Those two enzymes are both very robust and work well together.
Thank u for the reply.
Yes I agree your comment on sequential Vs Double digestion.
In pGADT7 vector there are about 27 bp between the EcoRI and BamH1 sites.
In my experiments:
For double digestion: I always run an aliquot of the double digest on the gel and after confirming the completion of digestion I used to go for the gel extraction of the remaining reaction mix.
For Sequential digestion:
I initially used 10 microgram of vector dna in 200 microlitres volume and 2 microlitres of enzyme. After running an aliquot of 5 microlitres on gel and confirming that it gave a single nice band (2 hours), I used the remaining of the digest for PCR purification. I didnt checked the concentration of this purified product.
Then I did digestion with this purified product in a 150 microlitre volume with BamH1(2 microlitres) and in parallel I digested the pGADT7 uncut plasmid with BamH1. For this I used 1 microgram of DNA. So for checking the completion of the digestion I used the vector digested with BamH1 alone. After confirming this I ran the digested vector (final product digested with EcoR1 first and BamH1 later) on .8% agarose gel. I gave a nice thick band. gel purification using qiaKit was done. The concentration of this final purified DNA was poor.
So is there any way to improve the method. I know I hav complicated it but I want a good vector to clone my inserts which is very essential for further progress.
Thank you for your pateince and kindly help me if you could!
okay from the sound of this it, appears that you are losing DNA at the column purification step. When you gel purify the DNA band is present and substantial. I am correct?
So... assuming you are using a qiagen DNA purification column
1 - always make sure that you add enough QG buffer to your gel. The DNA only sticks to the column due to the action of this buffer. If your gel slab is big it will dilute the QG buffer as the gel slab has a lot of volume. Excess Qg buffer is okay
2- Since pGADT7 plasmid is big, remember to add isopropanol to the gel+QG buffer mix. this will help the DNA bind to the column
3- pass the QG buffer + DNA solution 3 times through the column. This gives the column more opportunity to bind to the DNA.
4- when you elute the DNA, use warm elution buffer (68 C). It also helps if you leave the elution buffer on the column for a 2 minutes before spinning it down. Also helps if you elute your DNA in two steps. So rather than once with 50ul, do the first elution with 20ul, then a second elution with 30ul.
Lastly, you don't need a lot of vector to actually build your plasmid. Plentiful is good, but you probably have enough vector DNA to work with. Just remember, you won't get any papers for building plasmids. Sometimes one can lose tract of things.
So... assuming you are using a qiagen DNA purification column
1 - always make sure that you add enough QG buffer to your gel. The DNA only sticks to the column due to the action of this buffer. If your gel slab is big it will dilute the QG buffer as the gel slab has a lot of volume. Excess Qg buffer is okay
2- Since pGADT7 plasmid is big, remember to add isopropanol to the gel+QG buffer mix. this will help the DNA bind to the column
3- pass the QG buffer + DNA solution 3 times through the column. This gives the column more opportunity to bind to the DNA.
4- when you elute the DNA, use warm elution buffer (68 C). It also helps if you leave the elution buffer on the column for a 2 minutes before spinning it down. Also helps if you elute your DNA in two steps. So rather than once with 50ul, do the first elution with 20ul, then a second elution with 30ul.
Lastly, you don't need a lot of vector to actually build your plasmid. Plentiful is good, but you probably have enough vector DNA to work with. Just remember, you won't get any papers for building plasmids. Sometimes one can lose tract of things.
It sounds great! Thank you for the useful tips. I will include these when I am doing the next vector digestion.
It is right that I dont need a lot of plasmid, but to start with a new vector in my hand I always wish to do with controls which consumes great amount of vector digest. So I always worry about the vector digest concentration.
thank you for the comments.