I am attempting to work up a PFGE protocol for a Gram Positive cocci that is of interest to our lab. However, I am still getting a lot of undigested material stuck up in the loading well. I originally assumed that the SmaI digest was incomplete but we discovered it was the concentration of Lysostaphin relative to the cell suspension. Once we increased the Lysostaphin, we started to see faint bands however now all we are getting is smearing! (see attached) plus a lot of DNA still stuck in the loading well. Does rough handling of the cell suspension or delay in embedding the cells in agarose cause smearing? Or is there something wrong with the electrophoresis tank temperature or buffer temperature and the DNA is degrading? Or buffer, tubing, tank contaminants? Thanks in advance.
I think Artur is probably correct but star activity where there is too much enzyme or too much glycerol added with the enzyme can sometimes look like this
because the DNA was destroyed; that problem due to defect of handling or wrong of keeping of samples
no problems with the smear if the concentration of DNA I'd high.Try to make digestion with restrictions enzymes and repeat PAGE if there no smear so your DNA is ok but if the smear Is still present after digestion so you have a problem in DNA idolation
Hi!
I think you have undigested genomic DNA.
How much Lysostaphin did you added? Usually, for plug preparation we use a bacterial suspension with an OD620nm=0.05, and a final concentration of Lysostaphin of 50ug/ml in the lysis buffer.
Another cause of inefficient digestion of genomic DNA could be the lack of activity of the enzime due to:
- Loss of activity of the enzyme (you can check if it is cutting with DNA restriction on an regular agarose gel).
- Lack of b-mercaptoethanol in preSmaI buffer.
-Ineficient washing of the agarose plugs. After the step of proteinaseK you should wash several times the plugs (6x). Remanent proteinasaK can inactivate the restriction enzyme SmaI.
Also, the mearing you get is due to degradation of the DNA, probably due to DNAses present in the reagents you use. You may consider to autoclave the buffers.
good luck!
Sabrina
Thanks for your advice. I have de-contaminated the tank and tubing, made sure everything is clean and sterile including all reagents.
The plugs were washed 6X. I agree, there is either insufficient lysis or RE digestion. I have re-run the same plugs using only 1/4 slice with 75 U of RE. Could this be causing star activity? I still think the cell suspension ratio to Lysostaphin is the problem. Can I put them back into more Lysostaphin and go from there? This is how we made the plugs:
i. resuspend pellet with EC buffer to McFarland 6. This is where I suspect the problem lies.
McFarland Standard No. 0.5 1 2 4 6 8
Absorbance @600nm 0.063 0.123 0.242 0.431 0.653 0.867
ii. Transfer 150 uL of the cell suspension into a sterile microcentrifuge tube. Add 7 uL of Lysostaphin (@ 1 mg/mL) followed immediately by 150 uL of 2.0% Certified PFGE agarose.
iii. Transfer the plugs to tubes containing 500 uL of EC buffer and 10 uL of lysostaphin (1 mg/mL). Incubate overnight at 37 oC.
As you said, the problem must be en excess of bacteria. The ratio of cell suspension/lysostaphin you added is high.
We prepare a suspension with an OD620nm = 0.05, then transfer 200 uL of the cell suspension into a sterile microcentrifuge tube and add 200 ul of agarose for preparing the plugs.
After that, plugs are transfered to tubes containing EC buffer with a final concentration of 50 ug/ml Lysostyaphin, 100 ug/ml lysozime, and 50ug/ml RNAseA.
I hope this can be usefull.
The reason I had such a high cell suspension was that my organism takes 5 days to grow and in the past I have only managed to get small amounts of DNA from it using a Qiagen DNA extraction kit (for sequencing). I was concerned about the organism being viable enough for DNA extraction. For PFGE - I remember trying a 0.5 McFarland in the past and I didn't seem to get enough DNA in the plug! However I will try it again using those lysis enzyme concentrations - that may have been the problem!
Do you think I can I treat those plugs again with lysostaphin, lysozyme and PK and then go from there? (If I only use a 1/4 slice of the plug).
Is the RNAse treatment important?
Thanks for your help!
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