Digestion Problem - Cloning (Feb/01/2006 )
I have to clone a DNA fragment of 775 bp with ECOR V AND HIND III IN pET 32.
Both are compatible in Buffer 2. But after my ligation and transformation i did not get any thing in my plates. If I have to digest it separately. Then first i digest it with Hind III buffer 2 and after heat inactivation run it on gel and elute it in 8 to 10 microlit and again digest it with ECOR V with BSA and after dephosphorylation again run gel and elute it i 8 to 10 micolit water and used in ligation.
Please guide me in my experiment and suggest me about control. I am new to this.
best regards
samita
I have a similar problem and I am digesting first with, in this case, HindIII, and after without purification with Not I to minimize DNA loose during purification of the first digest.
What ratio vector/insert are you considering for your ligation'? are you sure your ligation buffer is working? as its better to make one time aliquots to avoid dATP decomposition
Both are compatible in Buffer 2. But after my ligation and transformation i did not get any thing in my plates. If I have to digest it separately. Then first i digest it with Hind III buffer 2 and after heat inactivation run it on gel and elute it in 8 to 10 microlit and again digest it with ECOR V with BSA and after dephosphorylation again run gel and elute it i 8 to 10 micolit water and used in ligation.
Please guide me in my experiment and suggest me about control. I am new to this.
best regards
samita
[quote name='tertu' date='Feb 1 2006, 06:32 AM' post='39424']
I have a similar problem and I am digesting first with, in this case, HindIII, and after without purification with Not I to minimize DNA loose during purification of the first digest.
What ratio vector/insert are you considering for your ligation'? are you sure your ligation buffer is working? as its better to make one time aliquots to avoid dATP decomposition
Did you have controlles the volume of glycerol respect the final volume of digetion? it is possible that its quantity it too much.
I am using buffer provided by supplier. I am using ECOR V AND HIND III OF NEB. AND BUFFER 2 OF NEB. MY REACTION IS AS
DNA = 8 ul. (pet 32) DNA isert = 8 ul.
ECOR V = 1 ul.
HIND III = 1 ul. HIND III = 1 ul.
Buffer 2 = 4 ul. Buffer 2 = 4 ul.
water = 21 ul. water = 29 ul.
BSA = 5 ul.
Total =40 ul. Total =40 ul.
temperature 37 c and its about 90 minutes.
and heat inactivation at 80 c and 20 minutes.
I did not add glycerol any where and ATP is in buffer.
suggest me where i am wrong
best regards
DNA = 8 ul. (pet 32) DNA isert = 8 ul.
ECOR V = 1 ul.
HIND III = 1 ul. HIND III = 1 ul.
Buffer 2 = 4 ul. Buffer 2 = 4 ul.
water = 21 ul. water = 29 ul.
BSA = 5 ul.
Total =40 ul. Total =40 ul.
temperature 37 c and its about 90 minutes.
and heat inactivation at 80 c and 20 minutes.
I did not add glycerol any where and ATP is in buffer.
suggest me where i am wrong
best regards
Result of my digestion is attached. 1kb DNA ladder and Two lanes of my digested vector and its comparison with undigested vector
I am going to ligate my insert digested with Hind III and Eco RV.
Reaction is as follows
Vector = 1ul
Insert = 3 ul.
T4 DNA ligase = 1ul.
10x Buffer= 1ul.
H2o = 4.
Lets see what happened this time.
Any suggestions and tips are welcome.
best reagrds
from
samita
I am using buffer provided by supplier. I am using ECOR V AND HIND III OF NEB. AND BUFFER 2 OF NEB. MY REACTION IS AS
DNA = 8 ul. (pet 32) DNA isert = 8 ul.
ECOR V = 1 ul.
HIND III = 1 ul. HIND III = 1 ul.
Buffer 2 = 4 ul. Buffer 2 = 4 ul.
water = 21 ul. water = 29 ul.
BSA = 5 ul.
Total =40 ul. Total =40 ul.
temperature 37 c and its about 90 minutes.
and heat inactivation at 80 c and 20 minutes.
I did not add glycerol any where and ATP is in buffer.
suggest me where i am wrong
best regards
Result of my digestion is attached. 1kb DNA ladder and Two lanes of my digested vector and its comparison with undigested vector
I am going to ligate my insert digested with Hind III and Eco RV.
Reaction is as follows
Vector = 1ul
Insert = 3 ul.
T4 DNA ligase = 1ul.
10x Buffer= 1ul.
H2o = 4.
Lets see what happened this time.
Any suggestions and tips are welcome.
best reagrds
from
samita
my digested vector and digested insert in gel.
But i have one more strange problem.
When i spin my transformation culture to make pallet (competent cell DH5 Alpha) the pallet is dark blue.
I am not able to understand any thing what happened at the last step. Please explain me what this.