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Team:Heidelberg/Templates/MM week22p
From 2013.igem.org
Contents |
2013-09-23
- sequences arrived: File:Heidelberg PIK10.1-pIK10.2-pIK10.3-pIK11.1-pIK11.2-2013-09-23.zip, File:Heidelberg PIK10.1-2013-09-23 VF2.clustal.txt, File:Heidelberg PIK10.1-2013-09-23 VR.clustal.txt, File:Heidelberg PIK10.2-2013-09-23 VF2.clustal.txt, File:Heidelberg PIK10.2-2013-09-23 VR.clustal.txt, File:Heidelberg PIK10.3-2013-09-23 VF2.clustal.txt, File:Heidelberg PIK10.3-2013-09-23 VR.clustal.txt, File:Heidelberg PIK11.1-2013-09-23 VF2.clustal.txt, File:Heidelberg PIK11.1-2013-09-23 VR.clustal.txt, File:Heidelberg PIK11.2-2013-09-23 VF2.clustal.txt, File:Heidelberg PIK11.2-2013-09-23 VR.clustal.txt
- sequences are right
- when pasting sequence of pIK10 into parts registry: realized that introduced XbaI site before BBa_B0030, can not submit part
2013-09-27
- need to remove XbaI site for submission (for Boston) -> pIK12: amplify pccB-accA2 with primer introducing SpeI, ligate SpeI+Xba+ sites
- run PCR of pIK8.6 with primers VF2+IK44 (3 ng DNA, 20 µl total volume, using Q5) -> f9:
| Cycles | temperature [°C] | Time [s] |
|---|---|---|
| 1 | 98 | 30 |
| 35 | 98 | 10 |
| 55 | 10 | |
| 72 | 180 | |
| 1 | 72 | 600 |
| 1 | 10 | inf |
- digest 867 ng pIK8.6 (3 µl of miniPrep from 2013-09-15) with EcoRI+XbaI (20 µl total volume, using 0.5 µl enzyme), treat with antarctic phosphatase (37°C, 60 min)
- gel-purify large pIK8.6 fragment, f9 (elute f9 in 17 µl)
2013-09-28
- digest f9 with EcoRI+SpeI (0.5 µl enzyme, 20 µl total volume), purify with Nucleotide Removal Kit
- ligate at RT for 1h:
| what | µl |
|---|---|
| pIK8.6 | 8 |
| f9 | 9 |
| T4 ligase | 1 µl |
| T4 ligase buffer | 2 µl |
- heat-inactivate at 75°C for 5 min
- transform 10 µl into TOP10, plate 10 µl, rest on Cm, grow at 37°C
2013-09-28
- lots of small colonies on both plates, several large colonies on both plates
- pick 4 small, 1 large colonies from 10 µl plate, grow in 2xYT+Cm at 37°C
