Template:Heidelberg/notebook/pc/week31
week number 31
▼2015-07-28 p415 digest
p415 BsaI and BamHI digest:
0.5 µl BsaI
0.5µl BamHI
1 µl DNA
2 µl Cut Smart
16 µl water
1h 37°C
▼2015-07-28 p415 PCR DpnI digest, DNA precipitation und trafo in e.coli
DpnI digest:
0.5 µl DpnI
4.5 µl Cut Smart
45 µl DNA (Q5 p415, Ph p415, Ve p415)
37°C 60min
DNA precipitation:
20µl NaAc 3M pH 5,5
10 min on ice
Centrifuge for 10 min at 4°C max
Pour the supernatant into a new tube
Add 2.5 volumes of 96% cold ethanol
10min on ice
Centrifuge max for 10 min at 4°C
Remove supernatant
Wash pellet with 500µl 70% ethanol
Dry pellet
Dissolve pellet in 10 µl sterile water
KCM Trafo
With p415 mutated BsaI side after PCR (Q5, Ph,Ve) DpnI degest and precipitation
▼2015-07-29 Chemical competent E. coli cells (CaCl method)
For Golden Gate Cloning preparation, one shot survival cells were made competent.
Chemical competent E. coli cells (CaCl method)
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• for ~ 200x 100 µl aliquotes
Materials
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• 100 mM CaCl2 (aq) - autoclaved
• Glycerol
Day 1
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• grow bacteria over night in 50 ml medium at 37°C
Day 2
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1) transfer 20-30 ml of the ON culture into 400 ml LB
2) grow at 37°C until an OD of 0.5-0.6 is reached (3-5 hours), put
CaCl_{2} on ice, prepare aliquoting buffer (20 ml CaCl_{2} with 10%
glycerol)
3) centrifuge bacteria for 30 min at 3750 rpm, 4°C
• *work on ice and as quick as possible from here on*
• ==> you will need 8 falcons (50 ml) in order to do so
4) discard supernatant, resuspend bacteria in 5 ml CaCl_{2} / falcon,
pool 2 falcons, add up to 50 ml CaCl_{2}
• ==> you should have 4 falcons from here on
5) leave on ice for 30 min
6) centrifuge for 20 min at 3750 rpm, 4°C
7) discard supernatant, resuspend in 10 ml aliquoting buffer for all 4
falcons, add another 10 ml aliquoting buffer
• *Do not vortex*
8) pipette 100 µl per eppendorf tube (pre-cooled on ice), put aliquots
in -80 °C freezer
Competence of E.coli were tested.
▼2015-07-31 p415-GPD BsaI mutation PCR
22.3µl water
10µl 5x buffer
2µl 50mM MgCl2
2x 2.5µl pimer
5µl 10mM dNTPs
5µl DMSO
0.3 µl DNA
0.5µl Velocity
25µl Q5 MM
2x 2.5 µl primer
0.3 µl DNA
5µl DMSO
14.7µl water
Program:
97°C 3min
97°C 30sec
55°C 30sec
72°C 4min
72°C 5min
1-3 25x
▼2015-08-01 Miniprep p415 for “general plasmids”
▼2015-08-01 p415-GDP open PCR
Durchführung
P415-GDP open PCR (to get ccdB inside later on)
Velocity:
10 µl 5x buffer
2 µl 50mM MgCl2
2x 2.5 µl primer (JD 24,25)
5 µl 10mM dNTP
0.2 DNA
0.5 Velocity
27.3 µl water
Q5
25 µl Q5
2x 2.5 µl primer
0.2 DNA
14.6 µl water
Program:
97°C 3min
97°C 30 sec
72/68.6/61.4°C 30 sec
72°C 4min
30x
72°C 5min
4 for ever
Gel extraction with Q5 61.4 samples for Gibson.
▼2015-08-01 Gibson
Gibson:
One with ccdBg(106.5ng/µl) and one with ccdBk(359ng/µl) both with p415 (12.5ng/µl)
Gibson ccdBg:
P415 6.62µl
ccdBg 0.39µl
water 2.99µl
Gibson MM 10µl
Gibson ccdBk:
P415 6.62 µl
ccdBk 1:2 0.23
water 3.14 µl
Gibson MM 3.14 µl
1h 50°C
▼2015-08-01 Yeast trafo with positive control construct
Method adapted from
Knop M, Siegers K, Pereira G, Zachariae W, Winsor B, Nasmyth KSchiebel E. Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines. Yeast, 15:963-972.
The transformation protocol was based on the LiOAc method (Schiestl & Gietz, 1989).
The following solutions were used:
SORB: 100 mM LiOAc, 10 mM Tris/HCl pH 8 (from 1 M stock), 1 mM EDTA/NaOH pH 8 (from 0.5 M stock), 1 M sorbitol (special grade for molecular biology from Merck), adjusted with diluted acetic acid to pH 8, sterile filtered (can be stored at room temperature for several months, a volume of 0.5 l was usually prepared).
PEG: 100 mM LiOAc, 10 mM Tris/HCl pH8 (from 1 M stock), 1 mM EDTA/NaOH (from 0.5 M stock) pH8 , 40% PEG3350 (Sigma), sterile filtered (can be stored at 4°C for several months, a volume of 50 to 100 ml was usually prepared).
Carrier DNA: Salmon sperm DNA (10 mg/ml, Gibco BRL, 2 kb average lenght) was boild for 10 min and cooled on ice. Such prepared carrier-DNA can be stored at -20°C and repeatedly used.
Growth of yeast cells:
Yeast cells were inoculated from a fresh pre-culture and grown for at least 6-8 hours or over night to a density of 1 to 2 OD600 at 30°C (approx. 4*107 cells per ml) in YPAD medium.
Competent yeast cells: Yeast cells were harvested by centrifugation (500 g, 5 min, room temperature), washed once with 0.1 to 0.5 vol sterile water (room temperature) and once with 0.1 to 0.2 vol SORB (room temperature). SORB was completely removed by aspiration. The cells were finally resuspended in a total volume of 360 ml SORB per 50 ml of cell culture and 40 ml of carrier DNA (0°C) was added. The cells were aliquoted into appropriate portions (e.g. 50 ml, at room temperature) and placed at -80°C (no shock freezing).
Transformation: Usually, 10 ml of competent cells were used for the transformation of plasmid DNA and 50 ml of cells for the transformation of a PCR product. The DNA was placed into a sterile 1.5 ml tube (maximal 2 ml plasmid DNA per 10 ml of cells), the competent cells were thawed and added. The suspension was mixed well before a 6 fold volume of PEG was added. Cells were mixed throughout and incubated at room temperature for approx. 30 min (up to 2 hours is no problem). DMSO was added (1/9 vol to make a final concentration of approx. 10%). The cells were placed in a water bath at 42°C for 5-20 min (15 min works best with most strains). The cells were sedimented (2-3 min at 2000 rpm), the supernatant was removed (since it slows down growth on the plates) and the cells were resuspended in 100 to 200 ml of liquid medium (YPD or selective medium, for kanMX6 selection see below).
Selection for transformants:
In cases were auxotrophy markers were used for selection of the plasmid or PCR product, cells were plated onto synthetic complete medium lacking the corresponding amino acid (His3MX6: SC-HIS plates; klTRP1: SC-TRP plates). In cases, where PCR-products containing the dominant resistance marker kanMX6 were used, the cells were resuspended in approx. 3 ml of YPAD, incubated on a shaker for 2-3 hours at 23 or 30˚C, harvested and spread on a G418 plate (further details are given in the text).
1µl DNA
10 µl cells
60 µl PEG
7.7 µl DMSO
▼2015-08-02 KCM Trafo with Gibson Assembly (see 2015-08-01)
ccdB resistant E. coli and top10 as control
▼2015-08-02 PCR to open p415 and p416 for ccdB
Velocity:
2µl buffer
2x 0.5µl pimer
1µl dNTP
1µl p415 1:10 or p416 1:10
0.2 Velocity
0/1/3 µl MgCl2
Add water to 10µl final volume
Q5
5µl Q5 MM
2µl GC buffer
1µl p415 1:10 or p416 1:10
2x 0.5µl primer
1µl water
Program:
97°C 3min
97°C 30 sec
67/63.9/59.6°C 30 sec
72°C 4min
30x
72°C 5min
4°C for ever
3x3 with ve, 3x with Q5, one p416ve and one p416Q5