User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/06/14

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Isolation of plasmid DNA

ABSTRACT

  • Purification of pBBRMCS5 and pSB1T3 plasmids with a standard protocol.

  • Our efforts of standardize the plasmid pBBRMCS5 were unsuccesful so a new strategy will be implemented. For details of this new strategy see the entry of my team partner Pablo at this link Plasmid standardization.

So the first step of this new strategy is again to isolate plasmid DNA. The two plasmids required are pBBRMCS5 and pSB1T3.

  • Melissa and I extract this DNA with the following protocol:
 1)Inoculate 3 ml of culture medium with the strain of interest and incubate for 12 hours at 300 rpm.
 2)Transfer 1.5 ml of the culture to an Eppendorf tube and centrifugate it at 14000 rpm for 3 min.
 3)Decant the supernatant and transfer the rest of the culture to the eppendorf tube used in the previous step.
 4)Repeat steps 2 and 3. 
 5)Add 1 ml of TE 10:! -1, and centrifugate at 14000 rpm for 3 min.
 6)Remove well the TE with a syringe. Be careful and don´t touch the pellet.
 7)Resuspend the pellet in 100 μL of Solution I (Glucose/Tris/EDTA) with help of a vortex.
 8)Put 200 μL of Solution II (NaOH 0.2 N/SDS 1%) and mix gently by inversion.
 9)Put 200 μL of Solution III (CH3COOK 5M,  pH=4.8) and mix gently by inversion 20 times.
 10)Centrifuge at 13000 rpm for 10 min.
 11)Transfer the supernatant to a new Eppendorf tube with help of a syringe.
 12)Add to this new tube 1 ml of ethanol at 100%. Mix with vortex and centrifuge at maximum velocity for 10 min.
 13)Remove the supernatant and add 1 ml of ethanol at 70%. Resuspend the pellet with help of the vortex.
 14)Centrifuge at 13000 rpm for 2 min.
 15)Remove the supernatant with a syringe.
 16)Dry the pellets in an speed vac for 5 min.
 17)Finally put 20μL TE-RNAse.

Melissa isolate pBBRMCS5 and I extracted pSB1T3. We the do an agarose gel for electrophoresis to see the products. The plasmid was not very clean.



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