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Team:DTU-Denmark/Notebook/27 June 2013
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Signal peptides on 4% gels 80V for 45 min. All other PCR-products on a 1% gel 100V for 45 min. | 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. | 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. | ||
| - | [[File:27.06.13_Sec_and_TAT_signalP.jpg|thumb|left|The gel of the successful PCR-amp. of the two signal peptides]] | + | [[File:27.06.13_Sec_and_TAT_signalP.jpg|thumb|left|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.]] |
Purification af the signal peptides where done with Illustra MicroSpin G-50 with filters all sequences under 50 bp. | Purification af the signal peptides where done with Illustra MicroSpin G-50 with filters all sequences under 50 bp. | ||
Revision as of 16:45, 30 June 2013
Contents |
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.
Conclusion from today
We have all PCR-fragments except the backbone.
Sec signal peptide can be amplified successfully with a [ramp|https://2013.igem.org/Team:DTU-Denmark/Methods/PCR-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.
