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Team:DTU-Denmark/Notebook/27 June 2013
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| + | {{:Team:DTU-Denmark/Templates/StartPage|27 June 2013}} | ||
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=208 lab= | =208 lab= | ||
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== Main purposes today == | == Main purposes today == | ||
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Run gels to verify [https://2013.igem.org/Team:DTU-Denmark/Notebook/26_June_2013 yesterdays] PCR-prodcuts. Make new PCR to amplify the signal peptides and the backbone. | Run gels to verify [https://2013.igem.org/Team:DTU-Denmark/Notebook/26_June_2013 yesterdays] PCR-prodcuts. Make new PCR to amplify the signal peptides and the backbone. | ||
| - | == | + | ==Who was in the lab== |
| + | <hr/> | ||
Gosia, Henrike, Kristian | Gosia, Henrike, Kristian | ||
==Procedure== | ==Procedure== | ||
| + | <hr/> | ||
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. | ||
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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. | ||
Gel purification of weak band of the GFP SF. | Gel purification of weak band of the GFP SF. | ||
| - | [[File:27.06.13_gel_puri_GFP_Sec_and_GFP_TAT.jpg|thumb|left|The gel from where we purified GFP SF Sec and GFP SF TAT]] | + | |
| + | [[File:27.06.13_gel_puri_GFP_Sec_and_GFP_TAT.jpg|thumb|290px|left|The gel from where we purified GFP SF Sec and GFP SF TAT]] | ||
| + | [[File:27.06.13_Sec_and_TAT_signalP.jpg|thumb|270px|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.]] | ||
== Conclusion from today == | == Conclusion from today == | ||
| + | <hr/> | ||
We have all PCR-fragments except the backbone. | We have all PCR-fragments except the backbone. | ||
Sec signal peptide can be amplified successfully with a [[Team:DTU-Denmark/Methods/PCR-ramp|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. | Sec signal peptide can be amplified successfully with a [[Team:DTU-Denmark/Methods/PCR-ramp|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. | ||
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| + | Navigate to the [[Team:DTU-Denmark/Notebook/26_June_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/28_June_2013|Next]] Entry | ||
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Latest revision as of 18:09, 30 September 2013
27 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 was 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 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.
Navigate to the Previous or the Next Entry
