208 lab

Main purposes today

Run gels to verify yesterdays PCR-prodcuts. Make new PCR to amplify the signal peptides and the backbone.

who were in the lab

Gosia, Henrike, Kristian

Procedure

Signal peptides on 4% gels 80V for 45 min. All other PCR-products on a 1% gel 100V for 45 min. Two new PCRs with ramp from 70°C → 60°C and 60°C → 50°C. Sec signal peptide on 70°C → 60°C and TAT signal peptide with new primer set 2 and 3 respectively on program 60°C → 50°C. Also backbone, GFPs and RFP where on these programs but non worked.

Purification af the signal peptides where done with Illustra MicroSpin G-50 with filters all sequences under 50 bp. Gel purification of weak band of the GFP SF.

The gel from where we purified GFP SF Sec and GFP SF TAT

The gel of the successful PCR-amp. of the two signal peptides. Going from left to right; 100bp ladder, Sec signalP, Sec signalP, TAT with TAT2 primers, TAT with TAT2 primers, TAT with TAT3 primers, TAT with TAT3 primers. Note that TAT with TAT2 primers are a little longer than TAT with TAT3 primers because the insertion of 2 additional amino acids.

Conclusion from today

We have all PCR-fragments except the backbone.

Sec signal peptide can be amplified successfully with a ramp 70°C → 60°C and TAT signal peptide with both TAT2 og and TAT3 primers can be amplified with ramp from 60°C → 50°C. Both with 0.1°C/s.