Template:Heidelberg/notebook/pc/week29

week number 29

▼2015-07-14 Yeast trafo

Because the protein construct could not be assembled via Gibson in E. coli a yeast transformation with the Gibson fragments was performed.
p413-GPD: 750ng (25µl)
GFP 500ng
Ubi 200ng
BFP 500ng
50µl competent yeast
300µl PEG
Add fragments and PEG to competent yeast
Incubate 40min at 42°C
Centrifuge at 500g for 3min
Remove supernatant
Resolve pellet in 100µl H20
Plate on –His plates 1:100 and rest

GFP fluorescence could be detected
 

▼2015-07-15 Gibson assembly of the protein construct after PCR purification of the DpnI digest

To increase the Gibson efficiency we purified our DpnI digest of the Backbone (p413-GPD) with the QIAGEN QIAquick PCR Purification Kit. Purification was performed according to manufacturers protocol. DNA was eluted with 30 µl H2O. (obtained concentration: 30.3 ng/µL)

With the insert fragments kept and the purified Backbone we performed a 20µL Gibson assembly reaction.

Solution	Amount
NEB Gibson Assembly Master Mix	10 µL
Backbone (p413-GPD)	2.18 µL
sfGFP (1:10 dilution)	2.91 µL
Ubiquitin (1:10 dilution)	2.06 µL
mTagBFP (1:10 dilution)	2.84 µL

 

Incubation: 1h at 50 °C

The reaction was transformed in aliquots of 0.5 µL and 2 µL into E.coli TOP10

Results:

On the plate transformed with 0.5 µL of Gibson reaction no colonies were observed. The plate with 2 µL of Gibson mix carried 3 colonies that went into colony PCR screening.

▼2015-07-15 Aussagekräftiger Titel

 

Equipment and reagents

TE: 10 mM Tris-HCl pH 7.5, 1 mM EDTA add 1.25µl of 10mg/ml Zymolyase and 875µl ß mercaptoethanol to 250µl TE before use

S buffer: 10 mM K2H PO4 pH 7.2, 10mM EDTA add 1.25µl of 10mg/ml Zymolyase and 875µl ß mercaptoethanol to 250µl S buffer before use

Lysis solution: 25mM Tris-HCl pH 7.5, 25 mM EDTA, 2.5% SDS

3 M natrium acetate pH 5.5

 

1. Scrape a large mass of yeast from a plate and resuspend it in 1ml of TE in a microcentrifuge tube (the OD600 of this suspension should be between two and five). Yeast from a fresh, two to three day-old plate work best. The yeast can also be obtained from a 2 ml overnight liquid culture.

2. Spin briefly to pellet the cells. Resuspend the yeast in 250µl of S buffer with Zymolyase and ß mercaptoethanol.

3. Incubate at 37°C for 30min

4. Add 55µl lysis solution. Vortex to mix.

5. Incubate at 65°C for 30min.

6. Add 83 µl 3 M sodium acetate. Chill on ice for 10 min

7. Spin in a microcentrifuge for 10 min. Pour the supernatant into a new tube.

8. Precipitate the DNA by adding 400µl cold ethanol. Incubate on ice for 10 min, spin for 10 min and pour off the supernatant.

9. Wash the pellet by centrifugation with 0.5 ml of 70% ethanol and dry the pellet.

10. Dissolve the pellet in 40 µl sterile water. Use 1-2 µl of this crude yeast mini-prep to transform E. coli by electroporation.