Team:Heidelberg/Templates/MM week22p

From 2013.igem.org

Latest revision as of 23:46, 4 October 2013

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

Lane 1: NEB 2-log; lane 3: pIK8.6 digested with EcoRI+XbaI; lane 5: PCR of pIK8.6 with primers VF2+IK44

• 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:

• 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:

• 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