Team:Marburg:Project:Notebook:Methods

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Notebook: Methods

Miniprep (Plasmid DNA Miniprep Kit II, Omega)
  • centrifuge at 10.000g for 1min
  • add 250µl Resuspension Buffer P1 per tube (incl. RNase), vortex
  • transfer suspension into new 1,5 ml tube
  • add 250µl Lysis Buffer P2 per tube, invert until lysate is clear (2-3min)
  • add 350µl Neutralisation Buffer N3 per tube
  • invert immediately (precipitate forms)
  • centrifuge 10min at 13.000 rpm
  • transfer supernatant onto column
  • centrifuge for 1min
  • add 500µl Washing Buffer PE (HBC Buffer) per tube
  • centrifugation for 1min throw filrate away
  • add 700µl DNA Wash Buffer per tube
  • centrifugation for 1min throw filrate away
  • add 30µl milipore per tube – let column sit for 2min
  • centrifuge for 2min
Gel Extraction (QIAquick Gel Extraction Kit)
  • band is cut out of the gel under UV light and transferred into a 1,5ml tube
  • weigh the gel with the tube
  • add QG (100mg gel → 100µl)
  • incubate for approx. 10min at 50-60°C, vortex to dissolve the gel
  • put spin colum into 2mlcollection tube
  • add dissolved gel onto column → centrifuge for 1min
  • discard flow-through
  • wash with 750µl Buffer PE → centrifuge for 1min
  • discard flow-through
  • place column in 1,5ml tube
  • elute with 50µl milipore water: incubate 5min at 37°C → centrifuge for 1min
Purification of PCR Products (QIAquick Gel Extraction Kit) short protocol

short protocol without gel extraction

  • add 3 volumes or QG Buffer to tubes and 1 volume isopropanol
  • place QIAquick spin column in 2ml collection tube
  • centrifuge for 1min →discard flow-through
  • add 750µl Buffer PE
  • centrifuge for 1min →discard flow-through
  • place column in 1,5 ml tube
  • eluate with 50µl milipore water
  • incubate for 5min at 37°C, centrifuge for 1min
PCR1
Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 1 min
5 Go To 2 30x
6 72 4min
7 8 infinite
PCR2
Step Temperature °C Time
1 95 2 min
2 95 20 sec
3 58 20 sec
4 68 2,5 min
5 Go To 2 40x
6 68 4min
7 8 Infinite
PCR3
Step Temperature °C Time
1 95 3 min
2 95 30 sec
3 58 20 sec
4 72 1 min
5 Go To 2 35x
6 72 5min
7 4 Infinite
PCR4
Step Temperature °C Time
1 95 5 min
2 95 30 sec
3 55-65 30 sec
4 72 2,5 min
5 Go To 2 35x
6 72 5min
7 4 Infinite
PCR5
Step Temperature °C Time
1 95 5 min
2 95 20 sec
3 55 20 sec
4 72 20 sec
5 Go To 2 35x
6 72 5min
7 4 Infinite
PCRQ5
Step Temperature °C Time
1 95 3 min
2 95 30 sec
3 55 30 sec
4 72 1 min
5 Go To 2 30x
6 72 5min
7 4 Infinite
PCRQ5-2
Step Temperature °C Time
1 95 5 min
2 95 30 sec
3 58 20 sec
4 72 50 sec
5 Go To 2 35x
6 72 5min
7 4 Infinite
PCR Q5 elongation for 2kb fragments
Step Temperature °C Time
1 95 3 min
2 95 30 sec
3 55 30 sec
4 72 2,5 min
5 Go To 2 35x
6 72 5min
7 4 Infinite
Taq PCR
Step Temperature °C Time
1 95 5 min
2 95 30 sec
3 58 20 sec
4 72 2,5 min
5 Go To 2 35x
6 72 5min
7 4 Infinite
Annealing with PNK Inactivation
Step Temperature °C Time
1 37 1h
2 65 20 min
3 95 5 min
4 95-4 1 min/1°C
5 4 Infinite
Medium
LB-medium Concentration Final
Ampicilin Ampicilin (1000x) 100µl:1000ml
Final: 50µg/ml
Canamycin Canamycin (1000x) 100µl:100ml Final: 50µg/ml
Chloramphenicol Chloramphenicol (1000x) 100µl:100ml
Buffer
Buffer Additions
-2.0 Buffer 2.0 (10x) + BSA (10mg/ml -10) → BSA end concentration 100µg/ml (0,1µg/µl)

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