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From 2010.igem.org

Tuesday

Contents 1 Qiagen miniprep: Plasmid extraction 2 Digest 3 Gel extraction 3.1 Agarose gel 3.2 Electrophoresis 3.3 Cut gel out 3.4 Gel extraction kit 3.5 Weighing 4 Set up ligation 5 Nanodrop

Qiagen miniprep: Plasmid extraction

The 5ml culture set up yesterday was pelleted down using the microcentrifuge, the pellet was resuspended in buffer P1. Lysis buffer P2 was used to release the DNA from the cell (this buffer contains RNAse to help reduce RNA contamination). PE buffer containing ethanol was used as a wash. We lysed the cells for 1 minute, gently inverting the tube. We neutalised the lysis with N3 buffer and centrifuged for 10minutes, only the plasmid DNA was left in suspension. The DNA was eluted at low salt concentration through a column membrane.

Digest

Cut with restriction enzyme EcoR1 and Pst1 1 μl of each. 10*buffer ... so 3 μl in 30μ1 therefore we can have 25μ1 of DNA Also no more than 10% Glycerol in the enzyme solution or the reaction would be inhibited.

Gel extraction

Agarose gel

Electrophoresis

Cut gel out

Gel extraction kit

Weighing

Set up ligation

Nanodrop