I've been trying to do the linearization of the pPICZalfaA plasmid. The Easy Select kit (Invitrogen) says to use the restriction enzymes PmeI, SacI or BstXI. My insert does not have restriction site for these enzymes. I have been trying with the PmeI and SacI (from NEB) with 6 microg of plasmid for 20U of enzyme. Also, I checked the reaction at 1h, 2h, 3h, 5h and 16h after incubation in 37°C. However, I obtained 3 bands in every linearizations that I tried... I don't know if this is correct, I think I should see only a single band, right? My questions are: How many bands should I see after the linearization? If I proceed to transformation, will the Pichia pastoris incorporate the plasmid and express the protein? Could anyone suggest a protocol for linearization with this enzymes?
I ran the uncut and the "cut" plasmid in two lanes, and I saw the uncut with single band and the cut with 3 bands and it ran less than the other one...
I tried with the enzymes separately...
Could anyone describe a protocol here? I want to compare with the one I am trying...
Thansks!
Dear fernanda,
after using these enzymes you must get only one band of 3.6 kb+ gene of interest...
As you told you are getting 3 bands. it seems your plasmid is not getting digested... it will be helpful if you can put your gel image with this question..
you should make sure that you are not using too much old restriction enzymes..
you can also check whether your restriction enzyme is digesting or not using digestion controls...
for me its looking your enzyme is not digesting cloned Plasmid..
For more efficient digestion you can use fast digest enzymes..
http://www.thermoscientificbio.com/restriction-and-modifying-enzymes/restriction-enzymes/fastdigest/enzymes
If possible try to Digest and linearize with PmeI enzyme to achieve more multicopy transformants.
You can
transform with uncut plasmid, but you will need to use 50–100 μg of DNA to
compensate for the 10 to 100-fold drop in transformation efficiency. However,
with selection on Zeocin, any transformants you obtain will probably contain
your construct. For best results:
Use electroporation to transform your cells
Use at least 50 μg plasmid DNA for each transformation
Add more information to get good responses..
all the best
Perhaps you are having a bad case of partial digestion? This is easy to tell by looking at the gel; please post an image. In any case, that should be solved by cleaning up your plasmid prep using any of the usual silica-based column kits.
Another possibility is loss of activity of the R.E. Check with a clean plasmid known to have PmeI, SacI or BstXI sites.
Yet another possibility is that what you think is pPICZa is actually another plasmid. Did you sequence the construct?
Hope it helps
Hello guys! I tested the PmeI in a new plasmid and it worked fine! So, the problem is not the enzyme.
I am sending the picture of gel and as you can see, the 3 bands.
Now, I ask to you, guys, maybe I am using a low quantity of enzyme, what do you think?
Thanks!!
It looks like partial digestion (or no digestion at all, but nicking of the plasmid due to residual nucleases). Complete digestions yield bands whose fluorescence decreases monotonically with decreasing molecular weight; in this case however (e.g. lane 2) the band with the lowest apparent MW (the one at the bottom) is the brightest. The enzyme/reagents are fine per your control PmeI reaction on another plasmid, so most likely the culprit is your prep. Either it is dirty, or the plasmid you are purifying is not what you think it is.
Did you sequence your construct? If the reads overextend into the vector, check that it is pPICZa indeed.
Yes, my plasmid was sequenced and has the insert...I also tested the enzyme in the circular pPICZaA.... and worked fine....
I think I will try the reaction with more enzyme... like 10U per 1 microg...
use FD enzymes for better digestion...
it seems you are facing incomplete digestion..
http://www.thermoscientificbio.com/restriction-and-modifying-enzymes/restriction-enzymes/fastdigest/enzymes
all the best
Guys, problem solved! I discovered that the miniprep quality wasn't very good... I expanded again the plasmid and everything worked fine! Thanks to all for the help and suggestions! Best regards!
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