Team:Goettingen/NoteBook w2

From 2013.igem.org

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Revision as of 07:05, 17 August 2013

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	∞

@@ Line 27: / Line 27: @@
 <tr><td>Buffer (5x GC buffer or 5x HF buffer)</td><td>10</td></tr>
 <tr><td>dNTP mix (12.5 mM each)</td><td>2</td></tr>
-<tr><td>Primer fwd iGEM_34 (5 pmol)</td>2<td></td></tr>
+<tr><td>Primer fwd iGEM_34 (5 pmol)</td><td>2</td></tr>
-<tr><td>Primer rev iGEM_35 (5 pmol)</td>2<td></td></tr>
+<tr><td>Primer rev iGEM_35 (5 pmol)</td><td>2</td></tr>
-<tr><td>Chromosomal DNA M. smegmatis</td>2<td></td></tr>
+<tr><td>Chromosomal DNA M. smegmatis</td><td>2</td></tr>
 <tr><td>DNA-Polymerase (Phusion or PhuS)<br /> or dH2O for water control</td><td>1</td></tr>
 <tr><td>dH2O</td><td>31</td></tr>