Team:Goettingen/NoteBook w1

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)

Backup plates:

Storage at 4 °C

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

Stored in red box at - 20°C

Primers arrived!

iGEM_32 ~ 37: dissolved in HPLC water, stored at -20^oC in our box

100uM stock , for PCR dilute 1:20 in HPLC water.

Primer 32 and 33 are strong in forming 2^nd structures, increase the amount in PCR

Media preparation:

1000ml LB+Ampicillin Agar => ca. 50 Plates (Black Code)

Transformation:

Colonies on the plate: parts 1- 7:

4ml LB with antibiotics, overnight culture for mini-prep[C1]

Backup plates: C1,C2,C3 for each part

Preparation of Antibiotic Stocks

1000x Ampicillin 10 1mL Stocks (Freezer red box)

1000x Chloramphenicol 10mL Stock (Falcon in Freezer)

Media preparation:

 250ml *4 Chl

 [use the ones marked with “5^th..6.13 LB^chl” on EVERY plate first. ]*

 250ml *1 Amp  (marked with black)

primer design<ordered>

Transformation:

Resuspended DNA from iGEM kit (already stored in red box in -20 frigde):