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From 2013.igem.org

June 10th, Monday

Cloning DarR ORF from g-DNA

Colonies on the plates: parts 8, 7 C1

10 ml LB medium with 10 μl antibiotics, overnight culture for mini-prep

backup plates: C1, C2, C3 for part 8

Colonies on the plates: parts 8, 7 C1

Preparation of 250 ml LB broth media (stored on shelf above bench)

10 ml LB medium with 10 μl antibiotics, overnight culture for mini-prep

backup plates: C1, C2, C3 for part 8

Re-streak of E.coli clones from prtas 6 and 7 on new LBChloram plates Preparation of new LBChloram plates (500 ml) and Preparation of 250 ml LB broth media (stored on shelf above bench) PCR with DarR primers with different enzymes and different buffers

• dilution of primer stocks 1:20 (100 pmol -> 5 pmol):95 μl HPLC water + 5 μl primer 100 pmol
• preparation of dNTP mix -> dilute stocks 1:8 (100 mM ? 12.5 mM):50μl dNTP + 200μl dH₂O
• diluted primers and dNTP mix stored in red box at -20 °C

1x reaction(50μl)

Component	Volume(μl)
Buffer (5x GC buffer or 5x HF buffer)	10
dNTP mix (12.5 mM each)	2
Primer fwd iGEM_34 (5 pmol)	2
Primer rev iGEM_35 (5 pmol)	2
Chromosomal DNA M. smegmatis	2
DNA-Polymerase (Phusion or PhuS) or dH2O for water control	1
dH2O	31

Master Mix for 6 reactions

Component	Volume(μl)
dNTP mix (12.5 mM each)	12
Primer fwd iGEM_34 (5 pmol)	12
Primer rev iGEM_35 (5 pmol)	12
dH2O	186

addition of template (chrom. DNA/dH2O), polymerase and buffer individually, then addition of 37 μl master mix

For tested combinations of buffer and Pol: see table for gel loading

PCR protocol (cycler 7; folder “Katrin” > “iGEM” > “DarR seq”)

Step	Temperature	Time
Initial denaturation	98.5 °C	5 min
Denaturation	98.5 °C	30 s
Annealing	60 °C (TA = TM (≈66 °C) – 6 °C)	35 s
Elongation	72 °C	2 min (Phu needs more time than Phusion!)
Final elongation	72 °C	10 min
Hold	15 °C	∞

• Protocol of 1 % Agarose-1xTAE gel
• 4 μl PCR reaction + 1 μl 5x DNA Loading Dye
• 3 μl 1 kb ladder (Quick Load)
• Run at 100 V in 1xTAE buffer
• Staining in EtBr and destaining in water
• UV detection

Loading scheme

M	1	2	3	4	5	M
1kb ladder (QuickLoad)	HF	GC	HF	GC	Water control with HF	1 kb ladder (QuickLoad)

Reactions stored at - 20°C in 50 ml Falcon