Double digestion - (Jun/29/2007 )
QUOTE (PUC18 @ Jul 1 2007, 05:52 PM)
QUOTE (scolix @ Jul 1 2007, 04:41 PM)
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
Hi
want to perform double digest using Sac1 from NEB and Pst1 from Bio Basic the buffer for Sac 1 is NEB1 and the buffer for Pst1 is the same as NEB1 except for the fact that TRIS HcL Concentration is 50mili Molar in place of 10 millMolar . Rest alll is same
Please tell me the best way to proceed ie cut first with Sac1 ant then purify fron gel then cut second
-genecat-
QUOTE (genecat @ Jul 4 2007, 06:02 AM)
Hi
want to perform double digest using Sac1 from NEB and Pst1 from Bio Basic the buffer for Sac 1 is NEB1 and the buffer for Pst1 is the same as NEB1 except for the fact that TRIS HcL Concentration is 50mili Molar in place of 10 millMolar . Rest alll is same
Please tell me the best way to proceed ie cut first with Sac1 ant then purify fron gel then cut second
want to perform double digest using Sac1 from NEB and Pst1 from Bio Basic the buffer for Sac 1 is NEB1 and the buffer for Pst1 is the same as NEB1 except for the fact that TRIS HcL Concentration is 50mili Molar in place of 10 millMolar . Rest alll is same
Please tell me the best way to proceed ie cut first with Sac1 ant then purify fron gel then cut second
Do separate digests and precipitate in between. You would have a buffer for the Pst1 from Biobasic so use that.
-scolix-
QUOTE (scolix @ Jul 4 2007, 06:45 AM)
QUOTE (genecat @ Jul 4 2007, 06:02 AM)
Hi
want to perform double digest using Sac1 from NEB and Pst1 from Bio Basic the buffer for Sac 1 is NEB1 and the buffer for Pst1 is the same as NEB1 except for the fact that TRIS HcL Concentration is 50mili Molar in place of 10 millMolar . Rest alll is same
Please tell me the best way to proceed ie cut first with Sac1 ant then purify fron gel then cut second
want to perform double digest using Sac1 from NEB and Pst1 from Bio Basic the buffer for Sac 1 is NEB1 and the buffer for Pst1 is the same as NEB1 except for the fact that TRIS HcL Concentration is 50mili Molar in place of 10 millMolar . Rest alll is same
Please tell me the best way to proceed ie cut first with Sac1 ant then purify fron gel then cut second
Do separate digests and precipitate in between. You would have a buffer for the Pst1 from Biobasic so use that.
thanks do i need to run a gel in between to check the purity
-genecat-
QUOTE (genecat @ Jul 5 2007, 12:02 AM)
QUOTE (scolix @ Jul 4 2007, 06:45 AM)
QUOTE (genecat @ Jul 4 2007, 06:02 AM)
Hi
want to perform double digest using Sac1 from NEB and Pst1 from Bio Basic the buffer for Sac 1 is NEB1 and the buffer for Pst1 is the same as NEB1 except for the fact that TRIS HcL Concentration is 50mili Molar in place of 10 millMolar . Rest alll is same
Please tell me the best way to proceed ie cut first with Sac1 ant then purify fron gel then cut second
want to perform double digest using Sac1 from NEB and Pst1 from Bio Basic the buffer for Sac 1 is NEB1 and the buffer for Pst1 is the same as NEB1 except for the fact that TRIS HcL Concentration is 50mili Molar in place of 10 millMolar . Rest alll is same
Please tell me the best way to proceed ie cut first with Sac1 ant then purify fron gel then cut second
Do separate digests and precipitate in between. You would have a buffer for the Pst1 from Biobasic so use that.
thanks do i need to run a gel in between to check the purity
run gel after the second digest to check if the digest worked and then purify it through a column.
-scolix-