"
Team:Goettingen/NoteBook w1
From 2013.igem.org
<img src="
" />
Plasmid mini-prep for Part1-7
Cryo-Stocks of E. coli transformants (C1, parts 1 – 7):
- 900 μl E.coli ON culture + 100 μl DMSO 100% in special tubes (ask Katrin or Katrin^^)
- vortex
- store at – 70 °C (red box)
Plasmid Mini-Preparation of parts 1 - 7:
ON cultures of C1 for parts 1 – 7 with kit “NucleoSpin? Plasmid” von Macherery-Nagel according to manual pp. 16-17 (NucleoSpin? Plasmid protocol for purification of high copy plasmids
Step 5: recommended washing of silica membrane with buffer AW was performed
Step 7: Elution with buffer AE pre-heated to 50 – 60 °C (in future: DON’T elute with this buffer, use pre-heated HPLC-H2O instead! No one knows what’s inside the buffer and its components (EDTA?) could interfere with sequencing and other reactions)
NanoDrop – Plasmid concentrations
| Part Number | c(DNA)[ng/μl] | A260/A280 | A260/A230 |
|---|---|---|---|
| 1 | 84.6 | 1.94 | 2.18 |
| 2 | 79.8 | 1.94 | 2.10 |
| 3 | 151.0 | 1.89 | 2.24 |
| 4 | 29.5 | 1.91 | 2.07 |
| 5 | 154.9 | 1.88 | 2.25 |
| 6 | 88.9 | 1.92 | 2.08 |
| 7 | 5.9 | 14.08 | 1.87 |
Stored in red box at - 20°C
Primers arrived!
iGEM_32 ~ 37: dissolved in HPLC water, stored at -20oC in our box
100uM stock , for PCR dilute 1:20 in HPLC water.
Primer 32 and 33 are strong in forming 2nd structures, increase the amount in PCR.
06th<img src="" />
Pick the colonies of part1-7
Media preparation
1000ml LB+Amp Solid medium => about 50 Plates(with black code)
Transformation
| Entry Numer | Location on the kit |
|---|---|
| BBa_B0034 | P5 2 M |
Pick the colonies of Part1-7
4ml LB with antibiotics, overnight culture for mini-prep[marked with C1]
Backup plates:marked with C1,C2,C3 for each part.
05th<img src="" />
Preparation of the medium, antibioticks, Transformation.
Preparation of Antibiotic Stocks
- 1000x Ampicillin 10 1mL Stocks (EPs in the red box in -20 freezer)
- 1000x Chloramphenicol 10mL Stock (Falcon in Freezer)
Media preparation
- 250ml*4 LB media with Cm
- 250ml*1 LB media with Amp
Primer design
| Code Name | Description |
|---|---|
| iGEM_36 | DarR operator sequence + biobrick prefix |
| iGEM_37 | DarR operator sequence + biobrick sufix |
Transformation
| Biobrick entry number | Mark |
|---|---|
| BBa_J23117 | part 1 |
| BBa_J23116 | part 2 |
| BBa_J23110 | part 3 |
| BBa_J23118 | part 4 |
| BBa_J61101 | part 5 |
| BBa_BE0240-Cm | part 6 -- Cm |
| BBa_B0015 | part 7 -- Cm |
| BBa_B0034 | part 8 |
Resuspended but not transformed
| Entry number | location on kit |
|---|---|
| BBa_E0204-Amp | P5 12 M |
| BBa_QO3121 | P5 20 N |
04th<img src="" />
Find the correct DNA sequence of DarR and primer design
| Code Name | Description |
|---|---|
| iGEM_32 | Forward primer for DarR ORF amplification, with biobrick prefix |
| iGEM_33 | Reverse primer for DarR ORF amplification, with biobrick sufix |
| iGEM_34 | Forward primer for DarR ORF PCR amplification sequencing. |
| iGEM_35 | Reverse primer for DarR ORF PCR amplification sequencing. |
