Trying to digest the pCAMBIA plasimd using BamHI and SacI which buffer should I use Is the Tangu buffer will work for both at the same reaction. my protocol is:
3ul Of each enzyme, 3ul of the buffer, 3ul of plasmid 10ng/ul and up to 30 ul neuclease free water.
but did not gaive any digestion, could someone help please?
NEB has a tool to help you design double digests:
https://nebcloner.neb.com/#!/redigest
Here's their suggestion for your enzymes:
https://nebcloner.neb.com/#!/protocol/re/sequential-heat/BamHI,SacI
Forgive me, but it reads as if you have a crazily large amount of enzyme present in the reaction, especially for 30ng (nanograms ) of plasmid DNA only.
Normally you should have a maximum of 10% of the reaction volume as added enzymes, otherwise the glycerol present in the storage buffer inhibits the reaction - which is perhaps what is happening in this instance.
Tango buffer is from Thermo / Fermentas if I'm not mistaken - it is analogous to NEB's CutSmart general restriction buffer.
They have a compatibility calculator similar to NEB's one mentioned above - it is at the following URL -
https://www.thermofisher.com/uk/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/restriction-enzymes-thermo-scientific/double-digest-calculator-thermo-scientific.html
nb - based on the calculator recommendation I have edited the protocol below to use 2 microlitres of SacI instead of 1 as originally written - this puts the total enzyme volume over 10 % of the reaction, but perhaps this is less critical with Thermo enzymes than NEB.
Try setting the digest up as follows -
Plasmid DNA 8 microlitres
MilliQ water 7 microlitres
Tango Buffer - 2 microlitres
Sac I - 2 microlitres
BamHI - 1 microlitre
Digest at 37 degrees C for 30 minutes. Compare with uncut vector to see a shift using 0.8% agarose electrophoresis.
Another possibility is that the DNA has been damaged during the alkaline lysis part of the mini prep : if the buffer 2 cell lysis step (NaOH / SDS) is incubated too long without neutralisation by buffer 3 (Sodium or Potassium Acetate), the DNA can be irreversibly knackered and will not digest properly.
Good luck - I'm sure you'll resolve this soon! Robin
Robin Dickinson thanks a lot Robin, T try to day a ratio semilar to the one you have sent Hope it will work. Many Thanks for your contribution.
Cheers
Mona
You could also try the full 15 microliters of DNA rather than DNA+water if your plasmid really is that dilute. My only reason for diluting is to reduce the risk of only part of the vector prep being digested, which could give you uncut carry-over in your subsequent cloning (to be avoided where possible!).
Robin Dickinson
Thank you for your ad, dose my plasmid need EtOH precipitation after that before transformation into E.Coli?
Er. Not quite certain I understand. If you try to transform plasmid that has been linearised with Sac / BamHI followed by gel purification you probably won't get very far. Are you performing a ligation after purifying the linearised pCAMBIA? I don't know exactly what you are doing, but if you give me a couple more details it sounds pretty straightforward otherwise.
Robin Dickinson
I try to digest both the plasmid and fragments withe BamHI and SacI to be able to ligate them during Ligation withT4 Ligase. after that I will transfere the ligated plasmied into E.coli to increase the number of plasmid copies. then miniprep then to AGLI to propagate the plasmid, then infect the plant with plasmid.
O.K ; that makes sense.
Digest vector and insert to completion - take care not to overload the vector on the gel (I often digest a slightly reduced amount and load it split into two or even three adjacent lanes on the gel so that the cut band is very clear - this reduces risk of carrying over single cut or uncut vector DNA). Once resolved, cut out the band and gel purify. Perform a typical T4 ligation with appropriate controls (I use NEB T4 ligase and their buffer without exception as in my hands it gives the best results), and transform ligation and controls to chemically competent DH5 alpha. Grow an appropriate number of colonies (if the controls are clean 2 should be enough, if there is small background, grow 3-4) and verify presence of insert by restriction digest. Sequence the plasmid if you think this is necessary.
Carry on with your procedure to propagate and infect.
With the ligation - take all the usual care with the buffer as this is almost as critical as the enzyme - thaw it slowly and put it away again as soon as it is used. NEVER vortex ligase buffer or reaction as this dramatically reduces efficiency. If necessary, set up a range of ratios of insert to vector, but for me usually at the first attempt I perform either 10u.l or 20u.l ligations - 7.5u.l of insert, 1u.l of vector, 1u.l of 10x buffer and 0.5u.l of enzyme, or 16/1/2/1 in 20u.l . I only vary from this if the ligations don't work first time or if there is limiting amounts of components.
Good luck!
Not at all - good luck with your project! If you need further assistance I do actually do this for a living .. :) keep an eye on my profile, announcements regarding my new enterprise coming soon.
right now, rdickinson at dcbiosciences.com ; another Email address will come along shortly but not ready yet.
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