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.