YG 2015 P6
Project 6: Preparation of library DNA and cloning of a
metabolic pathway mutation
In this project, we will try to clone a mutation in the yeast methionine
biosynthesis pathway by library complementation – hence, we need a yeast genomic
DNA library. Preparation of this library is not strictly “Yeast Genetics”, but it employs
some generally useful techniques and concepts.
There are two libraries we will be working with. One of them is a library that was
constructed in Joe Heitman’s lab at Duke University in the 1990’s. My colleague Dr.
Steve Hanes at the Wadsworth center of SUNY at Albany sent me the library in form of a
DNA pellet, and we have cloned quite a few genes from it. Unfortunately, the average
size of the genomic insert is rather small, so large genes are probably underrepresented.
The yeast community is generally very open when it comes to sharing materials
(plasmids, strains, libraries and sometimes even unpublished results), which in many
cases has made life much easier for scientists. Without a doubt, this culture of sharing has
contributed to the success of the yeast genetics field.
The other library was made in the early 1990s in France by Dr. François Lacroute. We
have used it extensively for cloning with great success.
Preparation of a library always brings with it the problem that members of the
library can (and will) be lost during each round of replication of the library. We once
tested a library that had been in my old lab for a long time and found that less than 50%
of the library plasmids (=”members”) had a genomic DNA insert. This means that when
you are trying to clone a gene, you will have to screen through twice as many
transformants as you would have to if all of the library members contained an insert
(which is, of course, never the case, but you can get close). It also means that some
inserts have been lost, and you may never be able to clone your gene of interest, because
it’s just not in the library.
Loss of plasmid members occurs during amplification of the library in E.coli.
Plasmids with large DNA inserts take longer to replicate, and consequently during
amplification of a library in E. coli, you will enrich the library for smaller plasmids (in
the worst case for empty plasmids). Some plasmids will carry genes that are detrimental
to E. coli and slow down growth severely, which can lead to loss of the plasmid in the
bacterial population because the cells carrying these growth-inhibiting plasmids will be
out-competed by other, faster growing cells.
For these reasons, it is not advisable to amplify libraries in a “batch” culture,
where E. coli cells transformed with the library DNA will be grown in one single flask.
Instead, the transformants are plated on selective plates at a density that allows the
formation of separate colonies. This way, each transformant grows on its own” microenvironment”, and competition for nutrients between transformants is not as fierce as it
would be in liquid culture.
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YG 2015 P6
Day 1 (Tuesday 1st week):
Electroporation (Electrotransformation) of Top 10 F’ E. coli cells (two groups
Heitman library; two groups Lacroute library)
Electroporation is a transformation procedure that yields in very high transformation
frequencies and works for both yeast and bacterial cells. Cells are made competent for
take up of DNA by electroporation by successive washes in destilled/deionized water( to
remove all the salts) and concentration of the cells. The competent cells are frozen at a
concentration of 1-3 x 1010 cells/ml in 10% glycerol and are stored at -70oC.
1. Set the BioRad Gene Pulser apparatus to 25F. Set the Pulse controller to 200.
Set the Gene Pulser apparatus to 2.5 kV (we are using 0.2 cm gap cuvettes)
2. Gently thaw out competent cells at room temperature and immediately put them
on ice (alternatively, just thaw out on ice). Place the cold sterile cuvettes on ice
(still in their pouches). Transfer 1ml of SOC media (rich growth media for
bacteria) into a labeled Eppendorff tube (“library transformation TOP 10”).
3. Add 2.0 l of library DNA (0.02 g/l DNA conc. in TE) to your tubes with
electrocompetent E. coli cells, mix well and let sit on ice for 30-60 sec. (The DNA
should be dissolved in water or a low ionic strength buffer like TE).
4. Take the cuvette out of the pouch, leave the sterile plastic pipette in the pouch,
but arrange it so you can take it out quickly. Make sure you do not touch the metal
electrodes of the cuvette with your fingers. Transfer the mixture of cells /DNA to
the cold electroporation cuvette and shake the cell suspension to the bottom by
slamming the cuvette on the table/bench.
5. Wipe off condensation on the cold electrodes with a paper towel and place the
cuvette into the chilled chamber slide (make sure the “nose” on the cuvette goes
into the gap of the slide). Push the slide into the chamber until the cuvette is
seated between the contacts in the base of the chamber.
6. Pulse at the above settings. You do this by pushing the two red buttons on the
gene pulser. After a few seconds, you will hear a beep from the Gene Pulser (it’s
not very loud, so pay attention!); when you hear it, immediately release the
buttons, swiftly take out the cuvette, add the SOC with the supplied sterile plastic
pipette, suck the SOC/cell mixture back out with the same pipette and transfer the
cells back into the tube that held the SOC. All this should happen fast (the quicker
the cells are in the SOC, the better the recovery. At this step, a few seconds matter
– if more than 10 seconds pass between the zapping of the cells and the transfer to
SOC, the transformation frequency goes down by 50%!!).
7. Incubate at 37oC for one hour, preferably in a shaker.
8. Spread the cells on 10 LB-Ampicillin plates (each group), according to the
instructions we will give you.
9. Incubate overnight at 37oC.
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YG 2015 P6
Day 2 (Wednesday 1st week):
Purification of the library DNA
I do not have any information on the complexity of the libraries (i.e. I don’t know how
many individual recombinants are in the library). However, I know the average insert
size of the Heitman library is about 5kb. How many individual recombinants should be
in the library to get a 99% probability that a gene of interest is represented in the
library?  Formula.
N=
Ln (1-P)
Ln (1-F)
N= number of library clones required
P= probability that the gene is in the
library
Average size of fragments
F=
Size of genome
We will talk about this during the Thursday lecture this week. Each group should
calculate the minimum number of library clones required by NEXT Tuesday.
To be reasonably sure that we will prep DNA from all possible library members, we will
make preparations from ~ 30-50,000 individual colonies.
1. Count the colony density (only one plate, count about ¼ to 1/8 of the plate and
multiply by 4 or 8, respectively).
2. Scraping the colonies off the plate: Add 5 ml of LB to a plate and used the glass
spreader to loosen the colonies from the agar by moving it over the surface of the
media in a circular motion. Especially smaller colonies can stick to the media, so
take care to also remove these. Do not, however, penetrate and take chunks out of
the agar
3. Pour the contents of the plates in two 50 ml Falcon tubes (2 x 25 ml cells in LB)
and mix well. Pool all cells in one Falcon tube and mix well (keep the empty
Falcon, see step 4.). Take out 2x 0.6 ml (we will use these to make duplicate
frozen stocks), add to cryotubes containing 0.6ml of 65% Glycerol, 0.1M MgSO4
and 25 mM TRIS pH 8.0 (3 tubes total) (this may vary depending on the
volume of our cryotubes; it is 0.9 ml + 0.9 ml if we’re using the 1.8 ml
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YG 2015 P6
tubes!); mix and freeze in liquid Nitrogen  -70oC freezer (Next time we need
more library, we can plate these cells straight on LBAmp and won’t have to do a
transformation)
4. Split the remaining cells again into two tubes (use the empt tube from step 3)
5. Spin down cells in Falcon tubes (4100x g for 15 minutes).
Alkaline plasmid maxiprep from bacteria
Solutions
Solution 1
50 mM Glucose
10 mM EDTA
25 mM TRIS HCl pH 8.0
Autoclave, store at room temperature
add 4 mg/ml lysozyme to several ml. Can be stored for several months at -20oC.
Solution 2
0.2 M NaOH
1% SDS
made fresh from 2 M NaOH and 20% SDS
Solution 3
60 ml 5 M postassium acetate
11.5 ml glacial acetic acid
28.5 ml H2O
Store at room temperature (do not autoclave). Before starting the procedure, put solution
3 on ice!
Method:
Add 6 ml of solution 1 to cell pellet and resuspend by vortexing vigorously. Make sure
there are no clumps of cells in the suspension - his is important for good lysis! Incubate at
room temp for 5 minutes.
Add 12 ml of solution 2. Mix gently by closing lid and inverting tube 3-4 times (do NOT
vortex!). Incubate on ice for 3-5 minutes.
Add 9ml of solution 3. Mix by vortexing vigorously. Incubate at room temperature for 5
minutes.
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YG 2015 P6
Centrifuge in tabletop at 4100g for 20 minutes at 4oC. Transfer supernatant into a fresh
falcon tube. Discard pellet.
The following extractions should be carried out at room temperature IN THE HOOD.
Add 22 ml of PCIA to the supernatant. Mix by inverting and vortexing vigorously.
Centrifuge at 4100g for 5 minutes.
Depending on how dirty the upper phase is, we may want to repeat this step:
Transfer the upper (aqueous) phase (with your DNA) in to a fresh tube and add 22 ml of
PCIA to supernatant. (Discard the lower phase into the Phenol/Chloroform waste).
Attention: the PCIA bottle contains two lipquid phases, an aequeous
phase (top phase) and the PCIA phase (lower phase).
DO NOT MIX THESE
Make sure you can see that both phases are well separated. Use the
liquid from the LOWER PHASE for the extraction (you have to pass
THROUGH the aqueous phase with your pipette tip)
Mix by inverting and vortexing vigorously. Centrifuge at 4100g for 5 minutes.
Transfer the upper phase to a fresh tube (discard the lower phase in Phenol/Chloroform
waste). Add 22 ml of CIA (Chloroform/Iso-amyl-alcohol). Spin for 2 min.
Transfer upper phase and split into TWO clean tubes (you will now have four tubes
altogether!). Discard lower phase (Phenol/ Chloroform waste). Add 24 ml of ice cold
abs. EtOH to EACH tube. Mix by inverting and several times. Incubate at room temp for
five minutes. Centrifuge at 4100g for 15 minutes. Pour off the supernatant and discard.
Add 10 ml of ice cold 70% EtOH to pellet and centrifuge for 2 minutes. Remove as much
supernatant as possible (discard the bulk supernatant in the sink – don’t lose your pellet
in the process! - and then use a micropipettor to get out the rest).
Air dry the pellets.
Dissolve each pellet in 100 ul of TE.
This type of DNA prep contains a lot of bacterial RNA. As we have found out that this
“dirty” prep gives us better transformation efficiencies, we usually do not clean it up. It
is, however, not so easy to determine the DNA concentrations in these library preps.
Restriction Digests:
For the Heitman library we will perform two restriction digests that will provide
us with some information on the quality of the library DNA prep.
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YG 2015 P6
1.) Digest with NotI restriction endonuclease. The NotI recognition site is eight base
pairs long. Therefore, the site is relatively rare (You do the math!). It is, however,
present in the plasmid backbone of our library plasmids (see map). Cutting with
this enzyme will linearize most of the library plasmids. Empty library plasmids
(i.e. without a genomic DNA insert) will run at the size of the plasmid backbone
in an agarose gel. Plasmids with genomic DNA will run higher in the gel. Using
this digest, we can estimate roughly the fraction of library plasmids carrying an
insert, as well as the approximate average insert size.
2.) A digest with NotI and XhoI will be a good control to run next to the Not1 digest
on an Agarose gel. The NotI and XhoI sites in the plasmid backbone are flanking
the insertion site for the genomic fragment. This digest will therefore produce a
linearized plasmid band and a smear of DNA representing genomic DNA
fragments cut by XhoI (and to a very small extent by NotI)
Perform both digests with 1 ul of a 1/10 dilution of library DNA in a total Volume of
20 l in NEB buffer 3 with added BSA (1ul of 10mg/ml stock). Use 10 units of each
enzyme and digest for 1.5 hours at 37oC. Transfer to freezer.
The Lacroute library will be cut with NotI, and SalI+NotI (NEB buffer3 +BSA).
Digest as above. Then transfer to freezer.
Day 3 (Tuesday 2nd week): Analyze digests/inoculate strain for
mutation cloning.
Add 1/10 of the digestion Volume of loading buffer and Run out the DNA on a 0.7%
agarose/TAE gel, along with a 1kb ladder marker. Run gel at 60 – 100 V until the
blue band from the buffer has traveled about 2/3 through the gel.
We know the relative amounts of DNA in the bands of the DNA size latter. This will help
us estimate the amount of plasmids with insert DNA versus empty plasmids.
Cloning of pathway mutations
We have several strains in our laboratory that carry well-defined marker mutations in
genes coding for enzymes involved in metabolic pathways like purine or pyrimidine (e.g.
uracil or adenine) base synthesis or amino acid biosynthesis (e.g. methionine, tryptophan
or leucine). We will use a methionine synthesis mutants in an effort to clone the wild type
genes using the DNA libraries you just amplified.
There are three major points in this practice:
1. First, you will learn a high-frequency yeast transformation method
2. You will get acquainted with the first steps of a simple cloning procedure using a
selection for yeast colonies carrying a library plasmid that contains a
complementing genomic fragment
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YG 2015 P6
3. This procedure will help us to determine the quality of the library preparations.
The better the library is, the higher the frequency of clones that contain the right
genomic fragment. The result (the ratio of positive clones vs. total number of
transformants) will give us (Hiltunen lab yeast geneticists) an idea of how many
tranformants we will have to screen with this library to clone the mutations we are
interested in.
Inoculate mutant strain:
BY4741 (Met-, Ura-) one pre-culture each group in 5ml YPD
Use good aseptic technique!
Day 4 (Wednesday 2nd week): high frequency yeast transformation
Perform the high frequency yeast transformation according to the procedure from
the Yeast Transformation Page of the Gietz lab. You can find it on the following website:
The yeast transformation home page (http://home.cc.umanitoba.ca/~gietz/). Select “The
best method”
We will both take an OD600 reading (dilute your overnight culture 1:200 in YPD; use
fresh YPD as the reference), and count the cells. The Haemocytometer described in the
Gietz procedure is a Neubauer type haemocytometer. More information on cell counting
will be given during class.
Each group will do a 10x transformation
We will use 1 l of library DNA per transformation. Because we are expecting our
target plasmid to be present at fairly low frequency (1 in 10,000?), the amounts of cells
we are plating will differ from the amounts given in the procedure:
Control plates (these are always –ura plates, because that is the marker gene on our
library plasmid backbones):
From one of your final cell suspensions (after step 12 in the Gietz Lab procedure,
see below), you will make dilutions (10-fold: 22 l + 200 l of H2O; 100-fold: 22 l 1:10
dilution + 200 l of H2O and 1000-fold: 20 l 1:100 dilution + 180 l of H2O). Plate
these dilutions on three separate SC –ura plates. These are our controls that will give us
an estimate of the transformation frequency, as all cells that have taken up a library
plasmid will be able to grow on this media.
Plates selecting for complementing plasmids:
Spin down the trafo mixes as in step 12, but resuspend the cell pellets in 200l
sterile H2O each and streak onto selective plates (SC – Met - Ura), 200 l per plate (10
plates total per group). Mark the plate on which you have streaked the cells from the
transformation that was also used to the control plates. Only cells that have taken up a
plasmid carrying the wild type gene of the mutant involved in the respective pathway
should be able to survive on this media. Grow for several days at 30oC.
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YG 2015 P6
Day 5 (Friday of the second week of the course):
Count the colonies on the SC –ura plates and determine the transformation
frequency. Usually, the transformation frequency is expressed as number of
transformants/g DNA. Because we don’t know the exact DNA concentration due to the
RNA left in the prep (and we don’t really care about it in this case), we will express it as
in transformants/l DNA. (Number of colonies on the plate times dilution factor: 10x,
100x, and 1000x for each respective plate).
Count the colonies on the other 9 selective plates (not the one that had the rest of
the prep used for the control plates) and divide this number by 9 to determine the
frequency of complemented cells per l of library DNA. Dividing the total
transformation frequency by this number will give us the number of transformants that
need to be screened to obtain a complementing clone.
Pick several colonies per group from the selective plate. Streak part of each
colony on selective plates (SCD- Met –Ura, SCD- Met, SCD - Ura) and a YPD plate
(streak for single colonies!). This is to verify that these colonies are really Met+, along
with two controls (positive – W1536 5B - and negative- BY4741).
Assistants check plates on Friday – transfer into fridge if reasonably-sized colonies
are visible. If not, check again on Monday!
Day 6 (Tuesday 3rd week):
Check the selective plates on which you have streaked your clones from the
selective plates. Inoculate four clones from the –Met, -Ura plate(s) into 10 ml SCD-Ura
each.
Grow O/N at 30oC in shaker.
Day 7 (Wednesday 3rd week)
To “rescue” the library plasmid from these complemented yeast cells, we will carry out a
yeast DNA miniprep with the cells from the overnight culture (this is the same procedure
as for the genomic DNA isolation in project 5)
Protocol for DNA Isolation from Yeast:
Charles S. Hoffman and Fred Winston, Gene, 57 (1987) 267-272
1. Harvest cells by centrifugation (4200xg, 5 min) and resuspend in 0.5 ml water,
transfer the cells to a 1.5 ml Eppendorf-tube and centrifuge again (13000 rpm, 2
min).
2. Decant the supernatant and vortex briefly the tube to resuspend the pellet in the
residual liquid.
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YG 2015 P6
3. Add 0.2 ml 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH=8.0, 1
mM EDTA. Add 0.2 ml phenol-chloroform-isoamyl alcohol (PCI) (25:24:1). Add
0.3 g acid-washed glass beads (0.45-0.52 nm diameter).
4. Vortex 3 to 4 min. Add 0.2 ml TE.
5. Centrifuge: 5 min, 13000 rpm. Transfer aqueous layer (with your DNA) to a new
Eppendorf-tube. Add 1.0 ml 100% Ethanol. Mix by inverting the tube.
6. Centrifuge ´: 2 min, 13000 rpm. Discard the supernatant and resuspend the pellet
(your DNA) in 0.4 ml TE plus 30 µg RNase. Incubate 5 min at 37C. Add 10 µl 4
M ammonium acetate and 1 ml 100% Ethanol. Mix by inverting the tube.
7. Centrifuge: 2 min, 13000 rpm. Dry the pellet (your DNA). Resuspend it in 50 µl
TE.
Use 2 µl of your yeast DNA miniprep for electroporation of E.coli Top10 cells. Streak
out on LB-Amp (100 l on one plate, 900 l (spin down cells and remove excess
supernatant) on the other) incubate o/n at 37oC.
We will take out the transformation plates for you the next day and transfer them to the
fridge. We will then inoculate four E. coli transformants for each group in LBAmp for
you next Monday)
Day 8 (Tuesday 4th week): Library plasmid preps, sequencing
We need to get the plasmids isolated, digested and analyzed on the gel before
noon, so we can have our sequencing samples ready before 13:00!
Make plasmid miniprep from E.coli overnight cultures (follow kit instructions) to obtain
the library plasmid. (Do NOT attempt to concentrate the DNA by precipitation if the
concentration is low!)
Measure concentration of DNA and digest 0.5 g with Not1/Xho1 (HeAl) or NotI/SalI
(LaCroute) (see preparation of library project).
Analyze digests on 0.7 % Agarose
If you have isolated a plasmid and the plasmid has an insert, we will sequence the insert.
Sequencing will be carried out at the Biocenter sequencing core facility. We will provide
them with plasmids and primer.
To one microfuge, add to a final volume of 6 ul:
150-300 ng of plasmid DNA
5 pmoles of sequencing primer (HeAl: #38, # 39; LaCroute: #40, #41)
sterile H2O
(4 sequencing samples each group)
We will put the tubes into a box in the coldroom. The tubes will be picked up by the
vahtimestari and brought to the DNA sequencing core facility
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YG 2015 P6
Day 9 (Wednesday 4th week):
Nothing to do – waiting for sequencing results
Day 10 (Friday 4th week): Sequence analysis
If we get good sequences, we will run a BLAST search against the
Saccharomyces cerevisiae genome database:
Identification of a cloned sequence in S. cerevisiae
In “my network places”, find the “Biochem” Network place
Open the “abi files” folder
Find the 2012 folder, search within for our sequencing runs
Find your sequencing run, open the notepad for the 5’ primer
In the notepad (if your sequence worked) select all and copy
Open your internet browser
Log on to the Saccharomyces genome database (SGD):
http://www.yeastgenome.org/
8. Find the “BLAST” link and click on it
9. Paste the sequence you copied from the notepad into the corresponding box
10. Also add a title in the corresponding box
11. Choose program BLASTN (usually is default)
12. Choose Complete genomic sequence, chromosomes (DNA) as database (usually
default)
13. Use Default settings
14. Click the “Run Blast” button
15. From the results, lick on the highest scoring link (lowest probability)
16. Note down the chromosome number and the coordinates of your sequence, then
click “ORF map”
17. You see a representation of the yeast chromosome you hit, with two dashed lines
representing the locus your sequence corresponds to.
18. Repeat from step 4. but this time choose your sequence for the 3’ primer
19. After you have identified the chromosomal locus corresponding to your 3’ primer
sequence, go to the SGD homepage
20. Click Gene/sequence resources
21. In “2. Pick a chromosome”, enter the chromosome number and the coordinates
for the sequences you have identified
22. Click “submit form”
23. Click “Chromosomal features map”
24. You see a representation of the yeast chromosome you hit. The dashed lines
indicate the location of your coordinates. Print out!
1.
2.
3.
4.
5.
6.
7.
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YG 2015 P6
For Project 2:
Into the “Quick Search” field, enter “pdr13”  search
On the ssz1 info summary page, click on the “SGD ORF” map
Examine the chromosomal location of PDR1. Do you notice a familiar gene?
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