Team:Ruperto Carola/Experiments

Protocols


LB-Medium

Chemical amount in [g/l]
yeastextrakt 5
pepton/ trypton 10
NaCl 10

Synthetic complete (SC)

Component Final concentration
Bacto yeast nitrogen base without amino acids 6.7 g/l
Amino-acid (dropout) mix 2 g/l
Glucose 2% (w/v)
Bacto-agar (for solid media agar plates) 20 g/l

Composition of HiFi PCR

Component Final concentration
HiFi-buffer x1
dNTPs 1 mM
MgCl2 2.5 mM (kanMX6) or 3.5 mM (natNT2)
Primer FWD or REV 100 ng
Betaine 0.5 M
HiFi-polymerase 0.5 M

HiFi PCR cycling parameters

#
Step Temperature Time
Initiation 97°C 3 sec
Denaturation 97°C 20 sec
Annealing 66°C30 sec
Elongation 72°C 45 sec/kb
Cycling: repeat steps 2-4 x29
Final elongation 72°C 5 min
Hold 4°C ∞

TAE

Reagent Volume Final concentration
1M Tris-Cl (pH 8.0) 1 nL 10 mM
0.5M EDTA (pH 8.0) 0.2 mL 1 mM
Distilled H2O 98.8 mL

LiSORB

Component Final concentration
LiOAc 100 mM
Tris HCl 10 mM
EDTA (pH 8.0 with NaOH) 1 mM
Sorbitol 1 M
Salmon sperm DNA (10 mg/ml) 4 mg/ml

LiPEG

Component Final concentration
LiOAc 100 mM
Tris HCl 10 mM
EDTA (pH 8.0 with NaOH) 1 mM
Polyethylene glycol (PEG) 3350 40%
PCR Restriction digest Yeast and bacterial transformation Gel electrophoresis

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Name F Name R Sequence F Sequence R
STE2 rev overhang 415 STE2 for overhang 415 agggaacaaagctggagctccaccgcggtggcggccgctctagaactagtggatccd accgggccccccctcgaggtcgacggtatcgataagcttgatatcgaattcctgcag
S3 knock-out S2 knock-out ACAGGAAAATGAGTCATTGAGATCGAAACTTTTCAACCTATCAATCAACAATCCCCGTACGCTGCAGGTCGAC AGAGAGTGACTGAGTGTTACATTAAATATATTTATATATAAACGTATGATATTTAATCGATGAATTCG
STE2 forBamh1 STE2 for Bam H1 TTTTTGGATCCATGTCTGATGCGGCTCCTTC TCGAGGGGGGGCCCGGTACCCTTATTATTATCTTCAGTCCAG
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You opened the first week and this last one, I guess? Great, so as you might know: this one is the week after wiki freeze, so the lab will transform into a boilerroom similiar to the sponsoring phase. Its selling time again. $$$