Team:Frankfurt/Protocol

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Team: iGEM Frankfurt - 2012.igem.org

Contents

Methods and Protocols

Plasmid Preparation

Plasmid Preparation from E.coli (Mini Preparation)

  1. transfer 1,5 ml of an overnight culture in a reaction tube
  2. centrifuge at 8000 rpm at RT for 5 min
  3. resuspend the pellet in 100 µl solution 1
  4. addition of 200 µl solution 2
  5. mix till the solution is clear
  6. incubate at RT for 5 min
  7. addition of 150 µl cold solution 3, mix till protein clumps are build
  8. incubate 10 min on ice, centrifuge 15 min with 10000 rpm
  9. transfer the supernatant in a clean reaction tube
  10. fill the reaction tube with 96 % EtOH, mix and let it precipitate at -20 °C for 10 min
  11. centrifuge 10 min at 10000 rpm
  12. wash the pellet with 70 % EtOH
  13. dry the pellet and resuspend it with 30 µl water or TE buffer


Solution 1

  • 50 mM glucose
  • 10 mM EDTA
  • 25 mM Tris-HCl pH8

Solution 2

  • 0,2 M NaOH
  • 1 % SDS

Solution 3

  • 3 M KaAc pH 5,5

Plasmid Preparation from Saccharomyces cerevisia

  1. overnight culture in 5 ml
  2. centrifuge 2 ml of the cells for 1-2 min
  3. wash the cells with water
  4. resuspend the pellet in 400 µl buffer 1 with RNase
  5. addition of 400 µl buffer 2 and mix carefully
  6. addition of 2/3 volume of glass beads
  7. cell destruction: vibrax the cellsin a 2 ml reaction tube for 5 min at 4°C
  8. transfer 500 µl of the supernatant in a clean reaction tube
  9. addition of 250 µl buffer 3, mix, incubate it for 10 min on ice
  10. centrifuge at 10000 rpm, 15 min
  11. transfer the supernatant in a clean reaction tube, fill with isopropanol and mix
  12. centrifuge at 13000 rpm, 30 min
  13. wash the pellet with cold 70 % EtOH and let it dry
  14. resuspend the DNA in 30 µl water or TE buffer


P1

  • 50 mM Tris/HCl pH 8
  • 10 mM EDTA
  • 100 µg/ml RNase A

P2

  • 0,2 M NaOH
  • 1 % SDS

P3

  • 3 M KAc

Transformation

Yeast Transformation

  1. Inoculate the synthetic complete medium (SC) with the strain
  2. Incubate with shaking overnight at 30°C
  3. Harvest the cells at a OD600 0,5-0,6 by centrifugation (3000x g, 5 min, RT)
  4. Wash the cells with 0,5 vol of sterile water (resuspend by shaking, centrifugate with 3000x g, 5 min, RT)
  5. Resuspend the cells in 0,01 vol of sterile water, transfer the suspension to a reaction tube and pellet the cells (3000x g, 5 min, RT)
  6. Resuspend the pellet in 0,01 vol of sterile filtrated FCC (frozen competent cell) solution
  7. Aliquot 50 µl of the solution into the reaction tubes
  8. Store the cells at -80°C for at least one night (up to one year)
  9. Mastermix for the FCC transformation mixture:
SubstanceVolume [µl]
PEG 3350 (50% (w/v))260
LiAcetat 1.0 M 36
Single-stranded carrier DNA (10 mg/ml) 10
Total volume 306
  1. Prepare DNA aliquots: Solute enough DNA (e.g. 100 ng plasmid) in 54 µl of water
  2. Unfreeze the cells in a 37°C block for 15-30 sec
  3. Centrifuge the solution at 13000x g for 2 minutes
  4. Remove the supernatant
  5. Add 306 µl of FCC transformation mixture to the cells
  6. Add 54 µl of the DNA to the solution and vortex shortly
  7. When all the reaction tubes are prepared, vortex the samples well until all pellets are completely resoluted
  8. Incubate the samples for 40 minutes at 42°C in a heating block
  9. Centrifuge the cells at 13000x g for 30 sec and pour off the supernatant
  10. Resuspend the cells in sterile water by vortexing
  11. Spread onto the appropriate medium
  12. Let the cells grow at 30°C

Yeast transformation and gap repair (example)

DNA FragmentSize[bp]Concentration[ng/µl]Equimolar Quantities°[ng]Used Volume[µl]
HMG-CoA16002242221
tHXT736051,6501
pPFK160054831,5
ERG2011001391521
tPFK236047,4501
pPGK160049831,7
GGPPS90014,51258,6
5520 765+ 19,8 µl linear p426 + 18,4 µl water

° determination of 50 ng for 360 bp
use of a vector-insert relation of 1:1
samples: linear p426 with 7 inserts

positive control (p426)
negative control (linear p426)

plating the samples on SCD-ura and incubation at 30° C

General procedure of a transformation via gap repair

  1. Amplification of the required DNA fragment with homologue ends of the plasmid (in our case homologue ends of the gene/promoter/terminator beside the DNA fragment) via PCR and an agarose gel to review the correct size
  2. Linearization of the plasmid and an agarose gel for review
  3. Yeast transformation with the PCR products and the linear plasmid (homologue recombination)
  4. Selection on an agar plate without the metabolite, which is on the plasmid for yeast selection (in our case it was uracil on p426 and histidin on p423)
  5. Inoculation of clones and isolation of the plasmids from yeast
  6. Transformation of a plasmid in E.coli and selection on LB medium with ampicillin (the plasmids have an ampicillin resistance)
  7. Isolation of the plasmids from E.coli
  8. Diagnostic restriction of the plasmid in order to find the correct plasmid with all inserts
  9. Transformation of the correct plasmid in yeast to do further experiments

E.coli Transformation

a) Production of competent cells

1. Inoculate 400 ml LB medium (pre-warmed to 37°C) in a 1 l flask with 100 µl of a fresh over night culture

2. Harvest the cells at a OD600 0,60-0,65 (in a room with 37°C, sterile)

3. Aliquot the solution in 8 x 50 ml falcons (sterile, pre-cooled on ice)

4. Incubate for 30 min on ice

5. Centrifuge for 12,5 min with 4000x g, 4°C

6. Resuspend the pellets in each 10 ml sterile water (4°C)

7. Centrifuge for 10 min with 4000x g, 4°C

8. Repeat steps 6 and 7 two times

9. Resuspend the cells in 10 ml 10% glycerine (+millipor, sterile, 4°C)

10. Centrifuge for 15 min with 4000x g, 4°C

11. Add 800 µl of 10% glycerine (sterile, 4°C) to the cells

12. Aliquot 40 µl of the solution in each reaction tube (pre-cooled) and freeze them in liquid nitrogen

13. Store them up to 6 months at -70°C


b) Transformation (on ice)

1. Add DNA (e.g. 1 µl of extracted yeast plasmids) to the frozen cells

2. Unfreeze the cells on ice

3. Fill the cells into cuvettes for electroporation

4. Electroporation (for 2 mm cuvettes: voltage I = 2,5 kV; resistance R = 2000 ohm; electrical current A = 25 µF; for 1 mm cuvettes: I = 2,2 kV; resistance R = 2000 ohm; electrical current A = 25 µF)

5. Fill the cells with 1 ml of SOC medium back in a reaction tube

6. Shake them for 1 hour at 37°C

7. Centrifuge for 1 min 8000x g

8. Decant the supernatant

9. Spread onto the appropriate medium

PCR

PCR protocol

ComponentsFinal ConcentrationVolume of Example PCR
Polymerase-buffer (e.g. for Phusion Polymerase)1x10 µl (5x)
dNTPs200 µM5 µl (2 mM)
Primer fw/rev respectively 0.2 µM respectively 1 µl (10 µM]
Template0.2 ng/µl1 µl (10 ng/µl)
DNA-Polymerase (e.g. Phusion Polymerase) 0.02 U/µl0.5 µl (2 U/µl)
Wateradd to 50 µl solution31 µl
Magnesiumchlorid 10 mM 0.5 µl (1M)
Total Volume 50 µl50 µl

Add all components with the right concentrations to a PCR tube. Pay attention to defrost phusion-polymerase on ice and add it at the very end. Put the PCR tube, containing a 50 µl reaction mixture, into a PCR cycler. Make the following settings for running the PCR programm:

PCR programm for one PCR run

StepsTemperatureTime
First Denaturation98 °C 30 sec
Denaturation98 °C10 sec
AnnealingDepending on Primer20-30 sec
Polymerization 72 °C 1 min/kbp for Tay-Polymerase; 20 sec/kbp for Phusion Polymerase
Final Polymerization 72 °C 5 min

Let the PCR-Programm run for about 35 cycles.

Restriction Digestion for Linearization of a Plasmid

Digest the choosen plasmid with one suitable restriction enzyme.

Example for digestion reaction:

ComponenConcentration
Shuffle Plasmid1 µl (3-5 µg/µl)
Restriction Enzymerespectively 1µl
Suitable Buffer 5 µl (10x)
Water add to 50 µl
Total Volume50 µl

Incubate the digestion mix 2 hours at 37 °C. AFter that purify the solution with e.g. a PCR purification kit.

Culture Media

Full Medium (YEPD) for Yeast
Yeast Extract1 % (weight/volume)
Pepton2 % (w/v)
Glucose2 % (w/v)
Synthetic Complete Medium (SC) for Yeast
Yeast Nitrogen Base0.17 % (w/v)
Ammoniumsulfate0.5 % (w/v)
Glucose2 % (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
Trypton2 % (w/v)
Yeast Extract0.5 % (w/v)
NaCl10 mM
KCl2,5 mM
MgCl210 mM
MgSO410 mM
Glucose20 mM

pH has to be regulated to pH=6.8-7.0

Full Medium (LB) for E.coli
Yeast Extract0.5 % (w/v)
Trypton1 % (w/v)
NaCl0.5 % (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

LBampicilline-Agar

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

LBchloramphenicol-Agar

Add 2 % agar to LB-medium. After autoclaving and cooling-down to 60 °C ethanolic chlorampenicol solvation is added.

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.

YEPDG418-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 puffer1x
Agarose1 % (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
EDTA18,6 g
Tris242g
Glacial Acetic Acid57,2 ml
Purified Water1000ml

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

Gels were run with a tension of 80-140 V.

Kits

PCR Purification Kit from Qiagen
Gel Extraction Kit from Qiagen
Midi Plasmid Preparation Kit from Qiagen

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