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Team:EPF-Lausanne/Notebook/October2011
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The elongation temperature was 68 C (as indicated by Roche pamphlet) for 3 minutes (for a 3 kb piece). The annealing temp is 52 C (as stated in Alessandro's sheet in the Dropbox). Vdog ran a touchdown PCR from 62 to 52 C) using 3 50 uL reactions. If all goes well, we should have 150 uL of K1 ready to go. I will run the gel tomorrow. | The elongation temperature was 68 C (as indicated by Roche pamphlet) for 3 minutes (for a 3 kb piece). The annealing temp is 52 C (as stated in Alessandro's sheet in the Dropbox). Vdog ran a touchdown PCR from 62 to 52 C) using 3 50 uL reactions. If all goes well, we should have 150 uL of K1 ready to go. I will run the gel tomorrow. | ||
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| + | == Thursday, October 13 2011 == | ||
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| + | Vincent ran the gel from yesterday's PCR, and there were three nice bands at the 2744 bp level. Alessandro's sheet in the Dropbox had predicted that amount (I mistakingly thought it was around 3 kb because I looked it up on the registry but the part we are amplifying is considerably shorter than the full plasmid). The PCR purification of the T7+Lysis and the K1 backbone went down perfectly even though the purification kit was out of spin columns (I had to use those from a plasmid mini prep kit). The average concentration was 100 ng/uL for the promoter+lysis constructs and 130 ng/uL for the K1 backbone. In the elution step, I used 30 uL instead of the prescribed 50 uL, since we wanted high concentrations. | ||
| + | |||
| + | The next step is to blunt the ends of both the plasmid backbone and the promoters+lysis constructs. For this, we used the T4 DNA polymerase from NEB and the associated protocol which I will add to the protocol section shortly (i.e. as soon as the TAs give me their approval). The technique seems straightforward but since the T4 polymerase has an exonuclease that reads from 3' to 5', the slightest mistake (i.e. overly elevated temperatures, excessive amounts of enzyme, not adding enough dNTPs, or letting the reaction run for too long) can all lead to recessed ends ... which would not be cool. | ||
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| + | Stay tuned. | ||
{{:Team:EPF-Lausanne/Templates/Footer}} | {{:Team:EPF-Lausanne/Templates/Footer}} | ||
Revision as of 15:46, 13 October 2011
Notebook: October 2011
Wednesday, October 12 2011
Over the weekend, Matt kindly made gene-specific lysis (36 uL). With Vincent's help for finding a convenient Tm calculator for single-nucleotide mutants, he made the extension PCR to add the 12 T7 promoters ahead of the lysis cassette. Vincent ran the gel for them and the results (thanks to Matt's insane Keynote skillz) are found below.
Vdog made a PCR to amplify the pSB3K1 backbone. Just so he doesn't forget, here's the protocol:
- 1 uL 10mM DNTP mix
- 10 uL buffer with MgCl2 (part of the Roche pack)
- .5 uL Polymerase (Expand Hifi regular)
- .5 uL Template (psB3K1 1:10. found in t7 promoters box -- the ligated version is in the ? box)
- 1 uL of each primer (50 uM)
- ddH20 to make 50 uL (36 uL if my count is right)
The elongation temperature was 68 C (as indicated by Roche pamphlet) for 3 minutes (for a 3 kb piece). The annealing temp is 52 C (as stated in Alessandro's sheet in the Dropbox). Vdog ran a touchdown PCR from 62 to 52 C) using 3 50 uL reactions. If all goes well, we should have 150 uL of K1 ready to go. I will run the gel tomorrow.
Thursday, October 13 2011
Vincent ran the gel from yesterday's PCR, and there were three nice bands at the 2744 bp level. Alessandro's sheet in the Dropbox had predicted that amount (I mistakingly thought it was around 3 kb because I looked it up on the registry but the part we are amplifying is considerably shorter than the full plasmid). The PCR purification of the T7+Lysis and the K1 backbone went down perfectly even though the purification kit was out of spin columns (I had to use those from a plasmid mini prep kit). The average concentration was 100 ng/uL for the promoter+lysis constructs and 130 ng/uL for the K1 backbone. In the elution step, I used 30 uL instead of the prescribed 50 uL, since we wanted high concentrations.
The next step is to blunt the ends of both the plasmid backbone and the promoters+lysis constructs. For this, we used the T4 DNA polymerase from NEB and the associated protocol which I will add to the protocol section shortly (i.e. as soon as the TAs give me their approval). The technique seems straightforward but since the T4 polymerase has an exonuclease that reads from 3' to 5', the slightest mistake (i.e. overly elevated temperatures, excessive amounts of enzyme, not adding enough dNTPs, or letting the reaction run for too long) can all lead to recessed ends ... which would not be cool.
Stay tuned.
