User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/06/23

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Plasmid isolation

ABSTRACT

  • Purification of pBBRMCS5 of BL21_DE3 cells.

  • Our efforts to standardize the pBBRMCS5 plasmid continue, so we change the strain of E. coli because DH5α is a DCM+ cell and the enzyme ApaI doesn´t cut if the DNA is methylated. Cells of the strain BL21_DE3 were transformed with pBBRMCS5 plasmid. The next step was to obtain this plasmid so Pablo and I performed the isolation process following the protocol of the next link : Plasmid isolation with the modification that steps 10, 12 and 14 were done at 4°C. We did an electrophoresis gel to see the quality of the plasmid and is shown in the following picture:


As you see the extractions are dirty with chromosome residues. So we can´t perform the digestion. We think that Solution III is not working properly so we are going to use another one for further isolations.

Determination of the DCM status of the new strain used

ABSTRACT

  • Experiment to verify if the new strain used is DCM+ or DCM-

  • The extraction of plasmid that we did before was not appropriate for the digestion with ApaI and SacI but it could be useful to see if the new strain is DCM-. So we performed a digestion with only ApaI (the enzyme sensitive to methylated DNA) to see if it can cut the DNA. The following proportions were used:
H2O 6μL
Buffer 4 10x 1μL
DNA 2μL
ApaI 1μL

We are going to wait till tomorrow to make an electrophoresis gel and find out if the enzyme ApaI cuts the DNA. If the product is digested then the strain is DCM- and we will continue using it.



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