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From 2013.igem.org

Contents 1 21-10-2013 1.1 Preparation of large plasmids 1.2 Restriction Digest of Preps with PvuI 1.3 Screening colonies of pSB4K5+DelH (without promotor) 1.4 Test if electrocompetent BL21-DE3 with DelRest and pIK8.6 contain DelRest 1.5 Screening ccdB-construct

21-10-2013

Preparation of large plasmids

6 ml of clones A1, A4, A7, B4

Restriction Digest of Preps with PvuI

0.5 µl PvuI-HF, 2 µl CutSmart, 1 µl 1:10 Prep (1-2 µg)
2 hours of incubation
expected bands: 11.6 kbp, 5.8 kbp, 4.6 kbp
--> only clone A1 was positive
--> A1 was send to sequencing with primer DN07

Screening colonies of pSB4K5+DelH (without promotor)

46 colonies
0.5 Primer (FS73 and FS95)
5 µl Dream-Taq
4 µl ddH₂O Results:

• Colonies B4, B9, B10 and C4 were positive
  
• Those colonies were inoculated in 6 ml LB Kan

Test if electrocompetent BL21-DE3 with DelRest and pIK8.6 contain DelRest

Screening of plasmid pFSN-D8w (positive control), Triple clone and electrocompetent BL21-DE3 with DelRest and pIK8.6
0.5 µl FS_14
0.5 µl VR
4 µl ddH₂O
5 µl Dream Taq
Results:

• All three screenings were positive. Consequently our electrocompetent BL21-DE3 with DelRest and pIK8.6 really contain DelRest.

Screening ccdB-construct

14 colonies were picked, we had a negative and a positive (pRB14) was used

Reagent	Volume
KH10	0.5 µl
VFII	0.5 µ
ddH2O	4 µl
Dream Taq	5 µl

Annealing temp.: 56°C, 35 cycles, elongation time= 50 s