Sc2.0 Megachunk
Assembly and Integration
Protocols
Ellis Lab
Ben Blount
30th June 2014
Table of Contents
Chunk Plasmid Preps .................................................... 3
Chunk Digests ............................................................... 4
Ethanol Precipitation and Ligation .............................. 5
Yeast Transformation ................................................... 6
Selecting for Integrants ................................................ 7
PCR Tag Analysis ........................................................... 8
Phenotypic Screening ................................................... 9
2
Chunk Plasmid Preps
Inoculate 2YT media, supplemented with the appropriate antibiotic, from the -80°C glycerol
stock.
Typical culture volumes in 50 ml Falcon tubes:
Chunk
n1
n2
n3
n4
n5
O/N Volume
25 ml
25 ml
15 ml
15 ml
10 ml
Volume P1
1.25 ml
1.25 ml
0.75 ml
0.75 ml
0.5 ml
Volume P2
1.25 ml
1.25 ml
0.75 ml
0.75 ml
0.5 ml
Volume N3
1.75 ml
1.75 ml
1.05 ml
1.05 ml
0.7 ml
Spin Columns
5
5
3
3
2
Incubate overnight in a shaking 30°C incubator.
Spin down tubes (ensuring they’re properly balanced) in the tabletop centrifuge at 4000
rpm for 15 minutes.
Resuspend pellets in P1.
Add P2 and mix by inversion.
Add N3 and immediately mix by inversion.
Transfer to microtubes (850 μl per tube).
Spin down at top speed for 10 minutes.
Pre-warm buffer EB to 55°C in heated block.
Transfer clear lysate to spin columns (750 μl per column).
Spin down at top speed for 1 minute.
Discard flow through and add 750 μl PE per tube.
Spin down at top speed for 1 minute.
Discard flow through and spin down at top speed for 1 minute.
Insert columns into fresh microtubes and apply 50 μl pre-warmed EB to membrane.
Incubate tubes at 55°C in heated block for 10 minutes.
Elute by spinning at top speed for 1 minute.
Determine plasmid yield by nanodrop.
3
Chunk Digests
Fill in a restriction digest data sheet and digest each of the chunks accordingly.
Aim to digest at least 30-40 μg of n1 and n2, 20-30 μg of n3 and n4 and 10-20 μg of n5
Incubate for 1 hour per required temperature
Load completed digests onto a 0.8% agarose gel and run at 116V for at least 40 minutes.
Ensure that bands are well separated and only excise the correctly sized bands.
Transfer bands to 2 ml microtubes, do not add more than 400 mg to each tube.
Add 3 times gel volume of buffer QG to each tube and incubate at 50°C until the agarose has
completely melted, vortexing every minute or so. Pre-warm EB to 50°C.
Once agarose has melted, add 1X original gel volume of isopropanol to the mix and vortex.
Using 1 spin column per microtube, add 750 μl sample to spin column and wait for 5
minutes.
Spin columns at top speed for 1 minute and then pour flow-through back into the top of the
column. Spin as before and discard flow through. Repeat process until all of the melted
agarose has been passed through the columns.
Add 750 μl PE per tube.
Spin down at top speed for 1 minute.
Discard flow through and spin down at top speed for 1 minute.
Insert columns into fresh microtubes and apply 50 μl pre-warmed EB to membrane.
Incubate tubes at 55°C in heated block for 10 minutes.
Elute by spinning at top speed for 1 minute. Add another 50 μl pre-warmed EB to the
membrane and repeat elution process.
Determine chunk yield by nanodrop.
4
Ethanol Precipitation and Ligation
Pool the chunks together with diminishing amounts of each subsequent chunk e.g. if you
have 10,000 ng of n1:
10,000 ng n1
5,000 ng n2
2,500 ng n3
1,000 ng n4
400 ng n5
Add dH2O to chunks to make up to 300 μl then add 30 μl of 3M NaAc pH5.2 and top up with
900 μl pre-chilled 100% ethanol (If chunk volume is greater than 300 μl, adjust other
volumes proportionately).
Incubate at -80°C for at least an hour.
Pre-chill microfuge to 4°C.
Spin down tube at 4°C, top speed for 30 minutes.
Carefully remove supernatant ensuring that pellet is retained.
Add 400 μl 70% ethanol and mix gently.
Spin down tube at RT, top speed for 10 minutes
Carefully remove supernatant and air-dry pellet until excess ethanol has evaporated. Be
careful not to over-dry the pellet.
Resuspend pellet in 10 μl 1X T4 ligase buffer. Add 0.5 μl T4 ligase and incubate at 16°C for at
least 18 hours.
Incubate at 65°C for 10 minutes to denature the ligase prior to transformation.
5
Yeast Transformation
**For best results, PEG and LiOAc solutions should be made up freshly for every
transformation**
Inoculate a 3 ml YPD kan50 culture with a colony of the strain to be transformed from a
freshly streaked plate. Incubate overnight shaking at 30°C.
The following morning, inoculate 2x 20 ml YPD cultures in 50 ml tubes with 200 μl each of
the overnight culture.
Incubate shaking at 30°C, periodically checking OD by adding 100 μl culture to 900 μl YPD in
a cuvette and measuring on the spectrophotometer.
Boil 50 μl of ssDNA for 8 minutes and then chill on ice.
When the OD is 0.5 +/- 0.1, harvest cells by spinning in the benchtop centrifuge at RT, 2000
rpm for 10 minutes.
Discard supernatant and resuspend each pellet in 10 ml 0.1M Lithium Acetate (LiOAc). Pool
the cells into one tube.
Spin down cells at RT, 2000 rpm for 10 minutes and resuspend cells in 400 μl 0.1M LiOAc.
Aliquot 4 x 100 μl into fresh microtubes. Two of these tubes will be megachunk
transformations, one will be the negative control (dH2O) and one the positive control (Predigested marker-swapper construct).
Vortex the ssDNA and add 10 μl to each tube. Vortex lightly, add 2 μl ligation/dH2O/markerswapper and then vortex lightly again.
Incubate for 30 minutes at room temperature.
Make up transformation mix:
Reagent
Final Concentration
50% PEG-3350
1M LiOAc
DMSO
dH2O
30%
0.1M
10%
N/A
Volume per
transformation
600 μl
90 μl
100 μl
98 μl
Volume for
master mix (5X)
3000 μl
450 μl
500 μl
490 μl
Vortex tubes lightly and add 888 μl transformation mix. Mix gently but well.
Incubate at room temperature for 30 minutes. Pre-heat heated block to 42°C.
Incubate cells at 42°C for 14 minutes, mixing gently by inversion halfway through.
6
Spin down cells at 8000 rpm for 2 minutes in a microfuge. Discard the supernatant by
aspiration with a pipette.
Resuspend cells in 1 ml 5mM CaCl2 and incubate at room temperature for exactly 10
minutes.
For each transformation, plate out 4x250 μl onto the appropriate selective medium. Also
plate out 250 μl of the positive and negative control reactions.
Incubate plates for 2 days in the 30°C incubator.
Selecting for Integrants
When colonies appear, count the colonies on transformation plates and ensure that the
control plates show the correct result.
Replica plate each transformation plates using a single velvet onto media selecting for the
lost marker and then onto media selecting for the gained marker. Store the transformation
plates at 4°C and incubate the replica plates at 30°C overnight.
Compare colonies on the lost marker selective plate to the original transformation plate. If a
colony is not present on the lost marker selective replica plate, ensure that it has grown on
the gained marker selective replica plate.
If a colony fits the selection criteria (e.g. for a gain of URA, loss of LEU selection the colony
grows on the original URA- and replica URA- plates but does not grow on the replica LEUplate) then streak the colony onto fresh selective plates and also inoculate a 3 ml selective
media overnight culture.
7
PCR Tag Analysis
Perform genomic prep on overnight cultures of candidate colonies.
PCR reactions are made up as follows:
2X GoTaq Green
F Primer (5 μM)
R Primer (5 μM)
gDNA
dH2O
5 μl
1 μl
1 μl
0.2 μl
2.8 μl
The PCR cycle (GoTaq Touchdown) is as follows:
20X
20X
95°C
95°C
70->50°C (-1°C per cycle)
72°C
95°C
55°C
72°C
72°C
4°C
5 minutes
45 seconds
45 seconds
1 minute
45 seconds
45 seconds
1 minute
5 minutes
∞
GoTaq Green PCR samples can be loaded directly into a gel without adding loading buffer.
When performing PCRTag analysis, always make sure that you include a BY4741 control for
every reaction.
It is recommended to initially screen a larger number of colonies for the WT and syn tags at
a single locus per chunk integrated and then thoroughly screen any colonies that pass this
screening round.
Phenotypic Screening
Inoculate 2 X 3 ml YPD cultures with a BY4741 colony and a colony to be assessed. Incubate
shaking at 30°C until the slowest growing culture reaches an OD of 0.5.
Add 100 μl of the OD 0.5 culture to a fresh microtube and add proportional amounts of the
other 2 cultures to fresh tubes along with DPBS to make the final mixtures 100 μl l of OD 0.5
culture.
Spin down cells at 8000 rpm for 2 minutes and resuspend pellets in 100 μl l DPBS. Repeat
centrifugation and resuspension in DPBS.
8
Serially dilute the cells so that you have at least 60 μl each of neat, x10-1, x10-2, x10-3, x10-4
and x10-5 cell dilutions.
Spot 10 μl of each dilution onto 3 x YPD (glucose) plates and 1 x YPEG (ethanol and glycerol)
plate as shown below:
Incubate:
1xYPD plate at 30°C for 2 days
1xYPD plate at 37°C for 2 days
1xYPD plate at room temperature for 3 days
1xYPEG plate at 30°C for 4-5 days
Following incubation, photograph plates in the gel dock. Compare the sizes of well defined
colonies for each strain (usually at the x10-4 dilution) using the contour and volume analysis
tools of the Quantity One software.
Perform a more thorough phenotypic screen with a wider variety of media and conditions
every 4 integration cycles or so.
9