Team:Panama/18 October 2010

October 18

Inoculation

We inoculated 5 colonies in liquid LB + amp medium

-3 ml medium + 1 colony

We incubated them overnight at 37C with shaking

Prepare other aliquot of primer for the amplification of the gene Rh1ab

Label the tubes well

Homemade Miniprep

5 samples

"Dirty" Mini Preps

P1 solution: 50mM Tris-HCl, pH8.0 10mM EDTA, pH8.0

P2 solution: 200mM NaOH, 1% SDS;

P3 solution: 3.0 M KAc, pH5.5

1. Spin down 1.5 ml cells at 14000 rpm for 2 minutes. Discard supernatant.

2. Resuspend in 300 ul Buffer P1 (with RNAse A)

3. Add 300 ul P2. Mix by inverting. Incubate at room temperature for 5 minutes

4. Add 300 ul of cold Buffer P3. Mix by inverting, incubate on ice for 5 minutes

5. Spin 14000rpm, 10 minutes

6. Transfer supernatant to a new tube. Extract with 500 ul Phenol:Chloroform:Isoamyl. Spin 14000rpm for 5 minutes

7. Transfer 800 ul of the aqueous phase to a new tube

8. Add 0.7 volumes of isopropanol (560 ul).Mix by inverting. Spin 14000rpm for 5 minutes at 4C

9. Remove isopropanol. Wash with EtOH 70%. Spin for 2-5 minutes at 14000rpm at 4C.Remove all EtOH.

10. Air dry. Resuspend in 20 ul TE Buffer.

Electrophoresis: Miniprep of plasmid +Rh1ab on a 1% agarose with 1x TBE

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Concentration of sample 5, according to Qubit fluorometer = 128ng/ul

Possible cause of the bands being so faint: We didn't let the pellet resuspend for an hour in step 10