Restriction Enzyme digestion - (Oct/08/2008 )
Is there a way to calculate the optimal time for HpaII digestion of 5ul PCR products? The PCR product is about 207 basepair long and after digestion, the fragment since should be about 100bp.
Incubation time is given at 37 degree celsius and using NEBuffer 1.. I tried it last week for 15 mins and there was nothing on the gel? my supervisor said its over digested.
-Allson_1987-
QUOTE (Allson_1987 @ Oct 9 2008, 11:37 AM)
Is there a way to calculate the optimal time for HpaII digestion of 5ul PCR products? The PCR product is about 207 basepair long and after digestion, the fragment since should be about 100bp.
Incubation time is given at 37 degree celsius and using NEBuffer 1.. I tried it last week for 15 mins and there was nothing on the gel? my supervisor said its over digested.
Incubation time is given at 37 degree celsius and using NEBuffer 1.. I tried it last week for 15 mins and there was nothing on the gel? my supervisor said its over digested.
Could you conform that your DNA do not degraded before digesting? or the content of your DNA is few?
stone
-stone757-
QUOTE (Allson_1987 @ Oct 8 2008, 07:37 PM)
Is there a way to calculate the optimal time for HpaII digestion of 5ul PCR products? The PCR product is about 207 basepair long and after digestion, the fragment since should be about 100bp.
Incubation time is given at 37 degree celsius and using NEBuffer 1.. I tried it last week for 15 mins and there was nothing on the gel? my supervisor said its over digested.
Incubation time is given at 37 degree celsius and using NEBuffer 1.. I tried it last week for 15 mins and there was nothing on the gel? my supervisor said its over digested.
15 min and having an overdigestion is quite odd. I experience overdigestion only when i do overnight once.
Well you could lessen the incubation time if you wish.
Run a control of uncut PCR product from the same sample/source u used to digest side by side when you run the gel.
do 1 min - 5min- 10 min ?
-Hanming86-
Anyone can enlighten me on the enzyme kinetic?
My HpaII is stated to be 50 000U/ml ,so if i use 1ul of it, it will contain 50units?
If i make a 10x dilution, it will contain 5 units?
my PI said that in theory, 1 unit is enough...
-Allson_1987-
QUOTE (Allson_1987 @ Oct 9 2008, 07:09 PM)
Anyone can enlighten me on the enzyme kinetic?
My HpaII is stated to be 50 000U/ml ,so if i use 1ul of it, it will contain 50units?
If i make a 10x dilution, it will contain 5 units?
my PI said that in theory, 1 unit is enough...
My HpaII is stated to be 50 000U/ml ,so if i use 1ul of it, it will contain 50units?
If i make a 10x dilution, it will contain 5 units?
my PI said that in theory, 1 unit is enough...
U could make a 1:10 dilution by diluting ur enzyme with 1x buffer.
1unit means can cut 1ug of linearized DNA under standard condition ( x minutes , y celcius, yada yada..)
Keep in mind that ur PI said " IN theory" sometimes in reality all these could be affected by amount of recognition sites in the sequence, the structure of the sequence ( accessibility issue) so on so forth .
Upload the gel picture if you willl. always good to have thing on "black and white" it helps alot!
-Hanming86-