Double digest and 3' end polishing with klenow - (Apr/23/2008 )

Dear all
I have this task to digest my vector (containing my insert) with SphI, do the end polishing (SphI leaves 3' overhang) and then cut the linearized, end polished vector with BamHI.
The protocol i have designed for this is as follows
3 ug DNA (3ul)
1.5 ul SphI (approx 8-10 u/ul)
SphI buffer 2 ul
water
Total reaction volume 20 ul 2 hours incubation at 37 C 5 ul for analysis on gel

15 ul left

1 ul Klenow
1 ul Klenow buffer (as im adding 10 ul more to the reaction volume without a purification step, the buffer already in the tube is sufficient for 15 ul so i will add for additional 10. What i know is that klenow is active in almost all RE buffers) I would rather like to know that should i add klenow buffer at this step or carry on with some additional SphI buffer?
dNTPs ? (this is where im a little confused. I have a stock of 5mM each dNTPs so can anybody tell me how much of it with how much final concentration of dNTPs i need to add) the protocols i have say they must be around 200uM-350uM


water

Total reaction Volume 25 ul incubation at room temperature (25 C) for 1 hour

Heat inactivation 75 C for 10 min

Directly adding BamHI and its buffer because interestingly the SphI buffer (takara) and BamHI buffer (NEB) have exactly the same composition and concentraions. Although BamHI (takara) and SphI (NEB) have different concentraions. I wonder why?

Starting Volume 25 ul
BamHI 1.5 ul
Buffer 1.5 ul (im adding 15 more ul to the reaction volume)
BSA x ul
water

Total reaction volume 40 ul incubate at 37 C for 2 hours

and finally gel purification


I want comments from some experienced fellas if there is any fault in this protocol or if i can do something to improve the efficiency of enzyme and yield of my DNA and also if anything can be done to further simplify my protocol

Hmmm....

• I would strongly suggest reducing the amount of restriction enzyme that is currently in use. The volume of restriction enzyme can not exceed 5% the volume of the restriction digest. The enzymes come in glycerol as protective buffer. However glycerol is also an inhibitor of enzyme activity. I given the volumes in use, reduce the volume of enzyme to 0.5ul.
  
• I would also suggest increasing the volume of your digest to at least 30ul. The amount of DNA digested is on the high side. If the DNA sample is not clean enough, this could lead to digestion problem, as dilution dilutes your problems away. You could also probably reduce digestion time to 1hr or 1hr 30minutes. 2hr is quite long.
  
• Klneow can live in NEB buffer 2 and NEB buffer 3. It likes most NEB buffer3. I am not sure what is the composition of SphI buffer provided by Takara. However if the digest is already in a buffer that Klenow can use, just add it in. there is no need to change buffer.
  
• According to NEB, SphI work in well in all buffer aside from buffer 3. I would suggest simply using NEB buffer 2. All enzyme present here (Klenow, BamHI) work in that buffer.
  
• M1V1 = M2V3, where M is molarity and V is volume.
  Given final volume is 25ul
  25u*0.2mM dNTP = 5mM*Xul,
  Xul = 1ul.
  Add 1ul of 5mM dNTP, and 25ul will have a 200uM of dNTP