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Revision as of 15:14, 14 September 2012 (view source) MS (Talk | contribs) (→Plasmid Preparation) ← Older edit		Revision as of 15:15, 14 September 2012 (view source) MS (Talk | contribs) (→Methods and Protocols) Newer edit →
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	===Yeast Transformation===		===Yeast Transformation===
	===E. coli Transformation===		===E. coli Transformation===
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		+	==PCR==
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	==Culture Medium==		==Culture Medium==
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Revision as of 15:15, 14 September 2012

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You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.

Contents 1 Methods and Protocols 1.1 Plasmid Preparation 1.1.1 Plasmid Preparation of E. coli (Mini Preparation) 1.1.2 Plasmid Preparation of Saccharomyces cerevisia 1.2 Transformation 1.2.1 Yeast Transformation 1.2.2 E. coli Transformation 1.3 PCR 1.4 Culture Medium 1.5 Agar Plate 1.5.1 LBampicillin-Agar 1.5.2 SCD-Agar 1.5.3 YEPDG418-Agar 1.6 Gel Electrophoresis 1.7 Kit

Methods and Protocols

Plasmid Preparation

Plasmid Preparation of E. coli (Mini Preparation)

Plasmid Preparation of Saccharomyces cerevisia

Transformation

Yeast Transformation

E. coli Transformation

PCR

Culture Medium

Full Medium (YEPD) for Yeast
Yeast Extract	1 % (weight/volume)
Pepton	2 % (w/v)
Glucose	2 % (w/v)

Synthetic Complete Medium (SC) for Yeast
Yeast Nitrogen Base	0.17 % (w/v)
Ammoniumsulfate	0.5 % (w/v)
Glucose	2 % (w/v)
Amino Acid Mix*	50 ml/l
Histidin**	0.25 mM
Tryptophan**	0.19 mM
Leucin**	0.35 mM
Uracil**	0.44 mM

pH has to be regulated with KOH to pH=6.3
!* contains no His, Leu, Trp and Uracil
** addition of this components depents on the respective selection medium

SOC-Medium for Regeneration of transformed Escherichia coli`s after Electroporation
Trypton	2 % (w/v)
Yeast Extract	0.5 % (w/v)
NaCl	10 mM
KCl	2,5 mM
MgCl2	10 mM
MgSO4	10 mM
Glucose	20 mM

pH has to be regulated to pH=6.8-7.0

Full Medium (LB) for E. coli
Yeast Extract	0.5 % (w/v)
Trypton	1 % (w/v)
NaCl	0.5 % (w/v)

pH has to be regulated with NaOH to pH=7.5

Every cluture medium has to be autoclaved to be sterile.

Agar Plate

LB_ampicillin-Agar

Add 2 % agar to LB-medium. After autoclaving and cooling-down to 60 °C steril ampicillin is added. Plates were poured.

SCD-Agar

Add 2 % agar to SCD-medium. After autoclaving and cooling-down steril amino acid solution is added. Dependent on the respective selective medium Histidin (0.25 mM), Trypthophan (0.19 mM), Uracil (0.44 mM) or Leucin (0.35 mM) are added. Plates were poured.

YEPD_G418-Agar

Add 2 % agar to YEPD-medium. After autoclaving and cooling-down sterile G418 (final concentration 2g/l) is added. Plates were poured.

Gel Electrophoresis

Agarose Gel (1x)
TAE puffer	1x
Agarose	1 % (w/v)

Solve agarose in TAE by boiling it. After cooling-down to 55-60 °C gel is poured.

TAE Puffer (50x) for Gel Electrophoresis
EDTA	18,6 g
Tris	242g
Glacial Acetic Acid	57,2 ml
Purified Water	1000ml

pH has to be regulated with glacial acetic acid to pH=8.

Kit

PCR Purification Kit
Gel Extraction Kit
Midi Plasmid Preparation Kit