Hi im doing double digest for pET22b. I used double digest of BamH1 and Hind111 for 1 hr at 37C. The content in (microliter) :
Plasmid=25
Buffer = 10
Bamh1= 1
Hind111= 1
AP=2 (alkaline phosphates)
do you mean 10ul of buffer. And which buffer. Buffers usually come in 10x concentration so there may be too much salt in the reaction mixture to cut properly . Also what buffer are you using as BAMH1 is fussy and likes it own buffer while ECOR1 is less fussy and cuts in most buffers to some extent although it can exhibit star activity and cut the wrong site if too much is used
Hi i used the yellow tango buffer. I checked it works well with BamH1 and Hind111. However i feel buffer hasnt been diluted well until 1 time. What do u think?
check the concentration of the buffer on the bottle or in the accompanying data sheet. If it is 10 times concentrated buffer and your final volume is 30ul use 3ul of buffer. If this does not work well cut for longer possibly even overnight for complete cutting
I feel your DNA concentration is low. Ideally the volume of DNA in reaction should be be within 10%(in your case it is >50%). We use Restriction enzyme assay buffer with 100mM NaCl for Hind III and EcoRI double digestion. Note that most of the enzymes require 2-5 fold excess units to cut circular substrate. You can add alkaline phosphatase at the end of the reaction, say after 1 hr and continue for 30 more minutes.
Tulasi,
If the photograph you have attached is of uncut and cut vector, it looks digested. There is mobility difference and there is no nicked form which is present in the uncut (and DNA is overloaded for this well size).
Well i have some issues with my plasmid extract. I seem to be getting a band 2 bands. One is the desired size and another looks like genomic DNA above 10kb. Any idea why?
you expect 2 or even more bands with uncut plasmid as the circular dna can fold and coill and supercoil into many different shapes and conformations which will appear to run at different sizes. When you cut this apparent mixture with a restriction enzyme it will be linearised and all of your bands will form one single band of linearised dna but post a picture as it could be bacterial genomic dna
Hi Paul ,
Thank you so very much , that actually helped to clear alot of my doubts. Now im going to double digest by pet22b, any suggestions on the suitable RE
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