Team:Panama/18 October 2010

From 2010.igem.org

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: 50mM Tris-HCl, pH8.0; P2: 200mM NaOH, 1% SDS; P3: 3.0M 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

• Photo

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