Team:Goettingen/NoteBook

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The beast and its Achilles heel:

A novel target to fight multi-resistant pathogenic bacteria

Timeline

June

07th

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

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

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

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.

Retrieved from "http://2013.igem.org/Team:Goettingen/NoteBook"

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