Restriction buffer - (Jun/05/2006 )
I am trying to do a restriction enzyme digest on my pcr product. The taq buffer (10X) contains 200 mM tris-HCl and 500 mM KCl.
The buffer for the enzyme contains 50 mM Tris-HCl (pH 8.0),10 mM MgCl2, 50 mM NaCl at 1X concentration.
My PI told me to add extra salt if needed and did not say anything about making the mixture with water, buffer and salt.
question: can i just add the dna,enzyme and salt (if needed) and do the digest?
or Do I need to make up the mix as given in some protocols?
How do I figure out if I need extra salt and how much?
Thank you!!!!
-maplegal-
please check this link
sometimes you can do the digest directly in the extension mix, depends on the enzyme
-aimikins-
QUOTE (maplegal @ Jun 6 2006, 06:24 AM)
I am trying to do a restriction enzyme digest on my pcr product. The taq buffer (10X) contains 200 mM tris-HCl and 500 mM KCl.
The buffer for the enzyme contains 50 mM Tris-HCl (pH 8.0),10 mM MgCl2, 50 mM NaCl at 1X concentration.
My PI told me to add extra salt if needed and did not say anything about making the mixture with water, buffer and salt.
question: can i just add the dna,enzyme and salt (if needed) and do the digest?
or Do I need to make up the mix as given in some protocols?
How do I figure out if I need extra salt and how much?
Thank you!!!!
The buffer for the enzyme contains 50 mM Tris-HCl (pH 8.0),10 mM MgCl2, 50 mM NaCl at 1X concentration.
My PI told me to add extra salt if needed and did not say anything about making the mixture with water, buffer and salt.
question: can i just add the dna,enzyme and salt (if needed) and do the digest?
or Do I need to make up the mix as given in some protocols?
How do I figure out if I need extra salt and how much?
Thank you!!!!
It looks as though your buffer is already pretty much right to go, apart from MgCl2. The 1x is 20 mM Tris (near enough to 50 for buffering), and 50 mM salt (enzymes don't really mind Na vs. K). Add some MgCl2 to 10 mM final from a high concentration stock (to reduce dilution effects, try 1% reaction volume of 1 M solution) add the enzyme and give it a try.
-swanny-
THank you so much. THe enzyme I am using is Hinf1.
QUOTE (swanny @ Jun 5 2006, 08:12 PM)
QUOTE (maplegal @ Jun 6 2006, 06:24 AM)
I am trying to do a restriction enzyme digest on my pcr product. The taq buffer (10X) contains 200 mM tris-HCl and 500 mM KCl.
The buffer for the enzyme contains 50 mM Tris-HCl (pH 8.0),10 mM MgCl2, 50 mM NaCl at 1X concentration.
My PI told me to add extra salt if needed and did not say anything about making the mixture with water, buffer and salt.
question: can i just add the dna,enzyme and salt (if needed) and do the digest?
or Do I need to make up the mix as given in some protocols?
How do I figure out if I need extra salt and how much?
Thank you!!!!
It looks as though your buffer is already pretty much right to go, apart from MgCl2. The 1x is 20 mM Tris (near enough to 50 for buffering), and 50 mM salt (enzymes don't really mind Na vs. K). Add some MgCl2 to 10 mM final from a high concentration stock (to reduce dilution effects, try 1% reaction volume of 1 M solution) add the enzyme and give it a try.
-maplegal-