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Team:Heidelberg/Templates/DelH week26
From 2013.igem.org
21-10-2013
Amplification of pFS_02 Backbone (HM_17 to FS_78; 4.1 kbp)
- Reaction
| Reagent | Amount [µl] |
|---|---|
| pSB6A1 | 1 |
| HM_17: (1/10) | 1 |
| FS_78: (1/10) | 1 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 35 | 98 | 1 |
| 58 | 5 | |
| 72 | 1:25 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Amplification of Backbone fragment for pFS_02 (HM_17 to FS_87; 4.2 kbp)
4 reactions with 20µl each, followed by DpnI digest
- Reaction
| Reagent | Amount [µl] |
|---|---|
| pSB6A1 | 0.2 |
| HM_17: (1/10) | 1 |
| FS_87: (1/10) | 1 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4.8 |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 1:25 | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 1:25 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Gibson assembly of pFS_02
| Reagent | Volume added (µl) |
|---|---|
| BB (HM_17-FS_78) | 0.24 |
| DN11-FS66 | 2.13 |
| FS_67-FS_68 | 2.89 |
| FS_69-FS_70 | 2.38 |
| FS_71-HM08 | 2.36 |
| Gibson Master Mix 2x | 10 |
Colony-PCR, screening for pFS_02
| Template | 96x 1 picked colony |
| Expected length [bp] | 521 |
| Named | 1A - 12H, DelH_pSB6A1_weak_promotor |
| Primer fw 10 µM | 96x 1 µl FS_73 |
| Primer rev 10 µM | 96x 1 µl FS_95 |
| One-Taq Polymerase (2x) | 96x 5 µl |
| ddH2O | 96x 4 µl |
| Cycles | Temperature [°C] | Time [s] |
|---|---|---|
| 1 | 95 | 120 |
| 12 | 95 | 30 |
| 68 (touchdown -0.5°C) | 30 | |
| 72 | 45 s | |
| 18 | 95 | 30 |
| 65 | 30 | |
| 72 | 45 | |
| 1 | 12 | inf |
10 colonies positive for screening none of them positive for restriction digest.
Preparation of large plasmids
6 ml of clones DelH+pSB4K5 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
Results:
--> 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 control 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
Results:
- All screened colonies were positive.
- Colonies 3, 7, 11 and 13 were inocculted
22-10-2013
Preparation of large plasmids
6 ml of clones pSB4K5+DelH B3, B9, B10, C4
Preparation of large plasmids
6 ml of clones ccdB 3, 7, 11, 13
