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Section 2 Molecular Genetics

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2.1 S. pombe plasmids o Yeast markers o Autonomous replication sequences (ars) o Expression vectors o The glucose repressible promoter 2.2 Transformations o Lithium Acetate procedure I (not used very often) o Lithium acetate procedure II: o Electroporation o Protoplast procedure. o Lithium Chloride procedure. 2.3 Integration of a plasmid into the genome. 2.4 Gene disruption and gene replacement. o Method I (not used as often now) o Method II: Gene replacement using PCR-based gene targetting plasmids containing ura4+ or kan module. o 2.5 Stability test. o 2.6 Cloning mutant alleles by gap repair. o 2.7 Recovering plasmids from S. pombe. 2.8 Multicopy overexpression of genes under the control of the nmt promoter

2.1 S. pombe plasmids

S. pombe plasmids consist of a bacterial origin of replication and selectable marker, a yeast selectable marker and an equivalent to an autonomous replication sequence (ars) which is responsible for high frequency of transformation.

Yeast markers
Budding yeast markers used in S. pombe are the LEU2 and URA3 genes. Plasmids containing these markers complement the S. pombe mutations leu1- and ura 4-. The URA3 gene is expressed very poorly in S. pombe and does not rescue the ura 4- mutation when it is present as a single copy or even at moderate levels. S. pombe markers commonly used are ura4+, sup3-5., leu1, his3, and his7.

Autonomous replication sequences (ars)

In contrast to S.cerevisiae, in S. pombe a bacterial plasmid such as pBR322 carrying a marker gene such as LEU2 is able to replicate often to high copy number. However, the transformation frequency obtained when using such plasmids is very low. The addition of S. pombe ars1+ sequences or the S.cerevisiae 2 m origin leads to high frequency of transformation and reduction in the copy number. So it seems that in S. pombe high frequency of transformation and effective replication capacity are to some extent independent phenomena.

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Plasmid vectors based on 2 m (pDB248, YEp13) are mitotically unstable, their copy number is low, they are much more prone to rearrangements (tandem duplications or deletions) and they are more difficult to recover from fission yeast than plasmids carrying S. pombe ars1+. Plasmids containing ars1+ are also very unstable (with the exception of pFL20 and pMB332); their copy number is higher and they tend to produce polymers with various numbers of repeats units. pFL20 and pMB332 yield rather stable transformants both mitotically and meiotically, due to the presence of a stb (stable) element . This element is not an ars sequence nor it is a centromeric sequence. Plasmids containing this element still segregated asymmetrically ten times more frequently during mitosis than S.cerevisiae CEN plasmids.

Expression vectors
Plasmids derived from the ones described above have been used to increase the expression of certain gene products. pSM1 and pSM2 are derivates from pDB248, made by inserting the SV40 early promoter. Genes linked to this promoter are expressed at moderate levels. pEVP11 contains the S. pombe adh+ promoter inserted into YEp13 S.cerevisiae vector. pART1 and pMB332 also have the S. pombe adh+ promoter inserted into pIRT2 and pFL20, respectively. The adh1 promoter is very active - about 5-20 x greater than the SV40 early promoter. pIRT2 is based on pUC118 and has S.pombe ars1 and S.cerevisiae LEU2 inserted. Recently, plasmids containing inducible promoters have been developed. These include various plasmids containing the thiamine repressible promoter, as developed by Maundrell (1990). Several versions are available in which the promoter sequences have been mutated to different degrees to give lower levels of expression (see Forsburg ref). pREP1 contains the wild type promoter. There is a significant background expression level ie with thiamine. The induced level is about 80X greater than the repressed level and about 6X greater than the level produced by the adh1 promoter. pREP41 has a 6X lower induced level and a 15X lower repressed level than the wildtype promoter. pREP81 has an induced level about 80X lower than the wildtype promoter (comparable to the repressed level of the wildtype promoter), whilst in the absence of thiamine the level is reduced a further 250 fold.

The glucose repressible promoter

Reference Hoffman & Winston 1989, Gene 84 473-479. This is a URA3 (S. cerevisiae) based vector pCHY21. It has the glucose repressible promoter of the fbp (fructose bisphosphase) gene upstream of a polylinker with 4 unique sites, Xho1, BamH1, PvuII and Nhe1. The promoter is OFF in 8% glucose and ON in 0.1% glucose +3% maltose.The vector has been tested with the cdc13?90 gene and worked well. All the cells became blocked in mitosis with a cut phenotype by 5 hours after shifting to 0.1% glucose 3% maltose. The average induction of fbp::lacZ is repressed derepressed 30 +/- 3 4405 +/-417 Intermediate expression can be obtained using b gal expression 3.0% maltose 523 +/-78 0.2% glucose 289 +/- 37

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0.5% glucose 88 +/- 12 1% glucose 55 +/- 6 Problems 1. Promoter is derepressed in stationary phase 2. URA3 is not well expressed when integrated in S. pombe 3. Wild type cells do not grow well in 0.1% glucose+3% maltose Method 1. Grow cells to an appropriate OD in minimal medium +8% glucose. 2. Wash the cells X2 with 0.1% glucose + 3% maltose minimal medium. 3. Resuspend the cells in 0.1% glucose + 3% maltose minimal medium and follow induction. The promoter is derepressed 30 fold by about 5h. 4. The cells should not be allowed to enter stationary phase

2.2 Transformations
S. pombe transformation is very straightforward and because most laboratory S. pombe strains are isogenic the frequencies are generally high regardless of the strain used. The lithium acetate method supposedly gives higher transformation frequencies although frequently far below the 10E6 reported; while electroporation is quick, convenient and gives higher transformation frequencies but is not always reproducible. Protoplasting is reproducible, gives a reasonably high transformation frequency but may not be so good for knocking out genes and is not frequently used; Lithium chloride is not used much due to the lower frequencies of transformation.

ithium Acetate procedure I (not used very often)

1. Grow 150 ml culture in MB medium to a density of 0.5 -1 x 107 cells/ml ( OD595 = 0.2-0.5). 2. Harvest the cells at 3000 rpm for 5 minutes at room temperature. 3. Wash cells in 40 ml of H2O? and spin them down as before. 4. Resuspend the cells at 1 x 109 cells/ml in 0.1 M lithium acetate (adjusted to pH 4.9 with acetic acid) and dispense 100 l aliquots into Eppendorf tubes. Incubate at 30C (25C for ts mutants) for 60 - 120 minutes. Cells will sediment at this stage. 5. Add 1 g of plasmid DNA in 15 l TE (pH 7.5) to each tube and mix by gentle vortexing, completely resuspending cells sedimented during the incubation. Do not allow the tubes to cool down at this stage. Add 290 l of 50 % (w/v) PEG 4000 prewarmed at 30C (25C for ts mutants). Mix by gentle vortexing and incubate at 30C (25C for ts mutants) for 60 minutes. 6. Heat shock at 43C for 15 minutes. Cool the tubes to room temperature for 10 minutes. 7. Centrifuge at 5000 rpm for 2 minutes in an Eppendorf centrifuge. Carefully remove the supernatant by aspiration. 8. Resuspend the cells in 1 ml of 1/2 YE5S? by pipetting up and down with a pipetman P1000.

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9. Transfer the suspension to a 50 ml flask and dilute with 9 ml of 1/2 YE5S?. Incubate with shaking at 32 C (25C for ts mutants) for 60 minutes or longer. 10. Plate aliquots of less than 0.3 ml onto minimal plates. If necessary, centrifuge the cells at this stage and resuspend in 1ml of media to spread more cells on a plate. Expected transformation frequency is 10E4 to 10E5 /g. Some strains may grow poorly or not at all in MB medium; URA 4 strains in combination with a temperature sensitive CDC allele seem to be particularly intractable. It is possible in some cases to overcome this by the following procedure: 1. From a preculture in YE5S? inoculate a 25ml culture of YE5S? and grow until in early exponential phase. 2. Harvest cells and resuspend in 200ml minimal medium (with appropriate supplements); grow again till early exponential phase. 3. Harvest cells and resuspend in MB medium for the last 1-2 cell divisions.

ithium acetate procedure II:

1. Grow fission yeast cells in MM to 1x107 cells/ml. 2. Pellet 50ml of cells per transformation. 3. Wash cells in 50ml sterile water. Transfer to eppis in 1ml water. Wash in 1ml of LiAc-TE. 4. Resuspend in LiAc-TE at 2x109 cells/ml (1/200 original volume). 5. Mix 100l cells with 2l carrier DNA at 10mg/ml and up to 10l of DNA; mix gently. 6. Incubate at RT for 10 min. 7. Add 260l of 40% PEG/LiAc-TE; mix gently. 8. Incubate 30-60 min at 29C-30C, or lower for temp. sensitive strains. 9. Add 43l pre-warmed DMSO; mix gently. 10. Heat shock at 42C for 5 min. 11. Pellet and wash once with 1ml water. 12. Pellet and resuspend in 500l water and plate 250l in duplicate. Solutions
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LiAc-TE: 0.1M lithium acetate, 10mM Tris pH 7.5, 1 mM EDTA Carrier DNA: boiled sperm DNA 10mg/ml LiAc-TE-PEG: LiAc?-TE plus 40% PEG4000

Electroporation
1. Grow cells to a density of 1 x 107/ml (OD595 = 0.5) in minimal medium. Transformation frequency is not harmed by growth until early stationary phase ( OD595 = 1.5 ) 2. Harvest cells by spinning at 3000 rpm (J 2-21) for 5 minutes at 20oC . Wash once by resuspending in ice-cold water and harvesting; a second time by resuspending in icecold 1M sorbitol and harvesting. 3. The final resuspension is in ice-cold 1M sorbitol at a density of 1 - 5 x 109 / ml. 4. 40-100 l of the cell suspension are added to chilled cuvettes containing the DNA for transformation ( 100 ng ) and incubated on ice for 5 minutes.

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5. Dry bottom of cuvette and pulse cells. The electroporator (Jensen) is set to 1.5 kV, 132 ?, 40 F. For a BioRad electroporator the settings are 1.5kV, 200 ?, 25F. 6. Cells and DNA are transferred to a pre-chilled cuvette and pulsed; 0.9 ml of ice-cold 1M sorbitol is then immediately added to the cuvette; the cell suspension is then returned to the Eppendorf and placed on ice while other electroporations are carried out. 7. Cells are plated as soon as possible onto minimal + sorbitol or minimal medium. Transformants appear in 4 - 6 days at 30oC Transformation efficiencies of between 10E5/g DNA and 10E6 /g DNA are expected. Note: This method does not seem to work very well with the sup 3-5 marker.

Protoplast procedure.
1. Grow 200 ml culture to OD595 of 0.2-0.5 (4 x 106-1 x 107 cells/ml) in minimal medium containing 0.5% glucose and supplements. 2. Harvest cells (at 2000rpm, 5 minutes, J-6), decant supernatant and resuspend the pellet in 10 ml of o 20mM Citrate/ phosphate pH 5.6 ( 2.82g/l Na2HPO4?, 4.2 g/l citric acid) o 40mM EDTA pH 8.0 3. Transfer to 50 ml round bottom tube, e.g. Oakridge tubes. 4. Harvest cells and resuspend each tube in 5 ml of o 50 mM Citrate/ Phosphate pH 5.6 ( 7.1 g/l Na2HPO4?, 11.5 g/l citric acid) o 1.2M Sorbitol. o Adjust to pH 5.6 with 5M NaOH and add 25 mg NovoZymTM 234 (added after autoclaving).  Incubate at 37C for 15-30 minutes (check under the microscope) until spheroplasts have formed. 5. Add 35 ml of o 10mM Tris-HCl pH 7.6 o 1.2M sorbitol o Divide between 2-4 round bottom tubes ( there should be no more than 3 x 108 spheroplasts/ tube). Spin gently at 2000 rpm for 5 minutes. 6. Wash twice more in 20 ml each time resuspending gently in 1ml first. At the last resuspension take a sample and count the number of protoplasts with a haemocytometer. 7. Resuspend gently first in 1ml then adjust to 2-5 x 10E8 protoplasts/ ml in: o 10mM Tris HCl pH 7.6 o 10mM CaCl2 o 1.2M Sorbitol o Combine the tubes. 8. Using 100 l protoplast/ transformation add 1-10 g of transforming plasmid in up to 1/10 total volume. Incubate at room temperature for up to 60 minutes. 9. Add 1ml of o 10mM Tris-HCl pH 7.6 o 10mM CaCl2 o 20% PEG 4000 o Incubate at room temperature for 15 minutes.

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10. Spin at 2000 rpm for 5 minutes, drain well and resuspend the protoplasts in 0.2-0.5 ml of: o 10mM Tris-HCl pH 7.6 o 10mM CaCl2 o 1.2M sorbitol o 0.5mg/ml Yeast extract, 5g/ml supplements ( leu, ura, ade, his) o Incubate at 30C for 30-60 minutes. 11. Plate out 0.2 ml aliquots onto well dried minimal sorbitol plates. Transformants appear in 2-5 days at 29-32C. Transformation frequency is about 1 x 10E4- 5 x 10E4 transformants/ g DNA. Protoplasts can be aliquotted out, stored at -70C in 10 mM Tris-HCl pH 7.6, 10mM CaCl2, 1.2 M sorbitol and used for at least 2 months. The frequency of transformation is 1 x103 transformants/g DNA for protoplasts stored in this way.

ithium Chloride procedure.

1. Grow 50 ml culture to stationary phase in YEPD medium ( 1% yeast extract, 2% peptone, 2% glucose) with shaking at 25-35C for 24-48 hours. 2. Use 10 ml of this culture to inoculate 40 ml of fresh YEPD medium and incubate for 4-5 hours. 3. Harvest cells at 3000 rpm for 5 minutes. Wash once in sterile distilled water and resuspend in 0.6 ml of buffer I (20 mM Tris-HCl pH 7.5, 2 mM EDTA, 0.2 M LiCl?) to give a total volume of about 1.2 ml and a final concentration of 2 x 109 cells/ml. 4. Incubate at 30C for 1 hour with gentle shaking. 5. In an Eppendorf tube mix: 200 l of competent yeast cells (4 x 108 cells) with 0.11g plasmid DNA and incubate at 30C for 30 minutes without shaking. 6. Add 700 l of buffer II (40% PEG 4000, 0.1M Li-Cl in TE buffer, sterilised with 0.2 m filters). Mix by inverting the tube gently. Incubate at 30C for 30 minutes. 7. Heat shock at 46C for 25 minutes. 8. Spread the cell suspension directly into YNB (0.67% yeast nitrogen base without aminoacids, 2% glucose, 1,5% agar). Colonies appear after four to six days at 30C. Transformation frequency is 4 x 103-9 x 103 transformants/g of plasmid DNA.

2.3 Integration of a plasmid into the genome.

In S. pombe integration by homologous recombination is usually more frequent than nonhomologous recombination; but for certain loci homologous recombination may only represent about 5-10% of the integration events. On average about 0.1% of the transformants obtained after transformation with an ars plasmid will have an integrated copy of the plasmid at the homologous locus. The frequency of integration can be enhanced up to ten fold by a single cut of the plasmid in the region of interest to facilitate the recombination event. There can be problems concerning the selective markers used that may complicate the integration of a plasmid in S. pombe. The S.cerevisiae URA3 gene on a multicopy plasmid complements S. pombe ura 4- mutations, but is poorly expressed in S. pombe and most of the integrated versions of URA3 fail to complement ura 4- mutations. Therefore LEU2 (does not always work), ura 4+, his3, leu1 or sup3-5 markers should be used. The latter marker is an

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opal nonsense suppressing tRNA gene which suppresses ade6-704. This marker has a deleterious effect for the cell when present in several copies. On minimal medium supplemented with 10 g/ml adenine or yeast extract medium ade6-704 mutant colonies are red but when suppressed by sup3-5 they are white. If a sup3-5 containing plasmid is not integrated into the genome then instability leads to cells lacking sup3-5 and hence to the formation of pink colonies. This contrasts with clones containing one copy of the integrated plasmid which are white, and enables a rapid distinction to be made between integrated and non integrated clones. Alternatively, ade6 itself can be transformed into an ade 6 deleted strain. For ura 4+ plasmids the best strain to use is ura 4-D18 that contains a complete deletion of the S. pombe ura 4+ gene and integration by homologous recombination at the ura 4 locus is thus avoided. Recently, the kanMX6 module has been widely used as a heterologous dominant marker that allows selection of G418-resistant cells in S.pombe (see Gene Disruption and Replacement Method II). To isolate an integrant: General procedure 1. Transform a yeast strain with the plasmid of interest. For example, linearize a leu1+ containing plasmid using a unique restriction enzyme site in the leu1+ gene (Keeney and Boeke (1994) Genetics, volume 136, page 849.) 2. Isolate a transformant colony and grow up in 100 ml of YES medium (i.e. non selective conditions) for about 20 generations (re-inoculate 1ml of this culture into 100 ml of fresh YES medium 2-3 times). 3. Plate out about 1000 cells/plate onto selective medium and incubate until colonies form. These colonies should be stable due to integration of the plasmid into the genome. This can be tested by replica plating to YES medium twice and then back to selective medium. 4. Confirm the integration by Southern blotting Procedure for sup3-5 1. Transform with the plasmid and plate out in the absence of adenine. 2. Replica plate to minmal plates with 10g / ml adenine. 3. After 2-3 days look for white fast growing colonies which are putative integrants. The pink colonies still have free plasmid.

2.4 Gene disruption and gene replacement.

Method I (not used as often now)
1. Make a disruption of the gene of interest by inserting the ura 4+ marker or the LEU2 marker gene in the ORF. If possible delete as much of the ORF as possible and conserve at least 1kb either side. 2. Purify this linear fragment. It is then possible to delete the native gene by integrating the marker flanked by flanking sequences of the gene of interest. This integration is performed by transformation of a diploid strain, as below. Homologous integration with a double crossover will result in excision of the native gene.

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3. Transform either a h-/h+ura 4-D18/ura 4-D18 ade6-M210/ade6-M216 or a h-/h+leu132/leu1-32/ ade6-M210/ade6-M216 diploid strain, depending on the marker used, with 100ng, 300ng, 1g of the fragment (less DNA is necessary if transforming using the Okayama or electroporation methods). Select diploid transformants expressing the ura 4+ or LEU2 gene. The transformant diploid can be sporulated in minimal glutamate or malt extract and the spores plated out to see whether the gene deleted is essential or not. If the gene is not essential the deletion strain can be maintained as a haploid. A non-sporulating diploid h+/h+ or h-/h- generated by endomitosis can also be used to make the deletion. This can be mated to a homozygous diploid of the opposite mating type to produce a tetraploid zygote which can sporulate and form four diploid spores. Some of these diploids will be heterozygous at the mating type locus and have one chromosome with a deleted copy of the gene of interest. These can be selected for and then sporulated to generate haploid spores which can be plated out as above. This approach is useful when it is necessary to analyse the effect of the deletion in different genetic backgrounds. Alternatively, h90 strains can be generated spontaneously from an h+/h+ diploid and identified by replica plating onto malt extract medium followed by iodine staining. Tetrad analysis can now be carried out using this strain. Gene replacements in S. pombe can be carried out using the same approach. A gene replacement event can be selected using a diploid strain in which one copy of the gene is disrupted with the ura 4+ gene, because ura 4- cells generated when the disrupted gene is replaced will be resistant to the drug 5-fluororotic acid (5-FOA). Thus, replacement of the ura 4+ disrupted gene with a linear DNA fragment containing the in vitro altered gene will convert the cells to a 5-FOA-resistant phenotype. It is necessary to maintain the presence of 5-FOA in the medium, as the effect is reversible. 5-FOA is used at 1 mg / ml in plates. NB this is an expensive chemical. It is always wise to check for the occurrence of the event by Southern blotting of the genomic DNA of the new construct.

Method II: Gene replacement using PCR-based gene targetting plasmids containing ura4+ or kan module.
This method is from J. Baehler and the reference for the transformation procedure is Keeney and Boeke (1994) Genetics, volume 136, page 849.

1. Design primers: 100mer oligos with 80nt to gene of interest and 20nt to ura4/kan module; primers should be HPLC purified. 2. Do 2-5 independent PCR reactions, pool reactions, purify the DNA by phenol/chloroform extraction and concentrate by ethanol precipitation. Resuspend in 10l of TE. 3. Follow lithium acetate method II (see section 2.1) for transforming PCR products, with the following modifications:

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o o o o o

Grow cells in YE5S? instead of MM. Plate on YE5S? with NO kanamycin. Incubate overnight at 30C. Replica plate the resultant lawn onto YE5S? with 100g/ml kanamycin. Incubate at 30C.

You will have large and small colonies- pick the large ones and restreak, etc.

2.5 Stability test.

This test is used to check the stability of a transformed plasmid. If the plasmid is replicating autonomously it will be lost in the absence of selection; on the other hand, if the plasmid has integrated or there has been a reversion or gene conversion event the phenotype is maintained after relaxing the selection. The procedure is as follow: 1. Take transformant colony and streak out to single colonies on YES agar with no selection for about 3 days until colonies form. 2. Replica plate to selective medium e.g. 35C on YES for a ts strain , 25C on minimal for the auxotrophic marker and check for the stability of the prototrophic (and / or other plasmid-borne) phenotypes. The sup3-5/ade6-704 system (described above) is particularly useful for this purpose.

2.6 Cloning mutant alleles by gap repair.

This technique is designed to clone chromosomal mutant alleles of previously cloned genes. 1. Construct a plasmid containing a selectable marker (LEU 2) and the wild type copy of the entire chromosomal region of interest. 2. Digest the plasmid with a restriction enzyme to completely remove the ORF. Purify the linear fragment containing the plasmid with the upstream and downstream flanking DNA sequences . 3. Transform 1 g of this fragment into the strain containing the allele of interest. Identify transformants expressing the selectable marker. The gap in the plasmid is repaired using the mutant chromosomal sequences as a template. 4. Recover the plasmid from yeast as described below. The efficiency of recovery of "repaired" plasmids versus "recircularised" plasmids appears to depend on the size of the flanking sequences, 1kb either side is recommended. On average 25 % of the plasmids obtained are "repaired". If overexpression of the mutant allele is deleterious to the cell it may not be possible to recover the repaired plasmid. An alternative procedure we have used involves integrating a sup3-5 containing plasmid adjacent to the mutant allele and then cutting out this plasmid with the mutant allele to recover it in E. coli. It is also simple to obtain the chromosomal mutations by PCR using the flanking sequences as oligonucleotide primers, although care is necessary since PCR is mutagenic (approx 3 x 10 -5/ base ).

2.7 Recovering plasmids from S. pombe.

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Plasmid recovery from S. pombe is difficult as 2 m based plasmids often seem to form multimers, and rearrangements (tandem duplication or deletions) are frequent. This problem can be avoided by using the plasmid pFL20 or derivatives that contain the stb element and remain as monomers. Furthermore, ars1 plasmids, which comprise many of those currently in use, do not tend to rearrange and are easy to recover. It is worth noting also that yeast material in the final preparation appears to inhibit E. coli transformation and thus using more of the preparation may not be a good idea To recover a plasmid from a transformant there are two procedures which are routinely used: Procedure A 1. Grow up 10 ml of cells under selective conditions to OD595= 1 ( 2 x 107 cells/ml). 2. Spin down the cells 3000 rpm 5 minutes. 3. Resuspend in 1.5 ml: o 50mM Citrate/ Phosphate pH 5.6 ( 7.1 g/l Na2HPO4?, 11.5 g/l citric acid) o 2M Sorbitol o Adjust to pH 5.6 with 5M NaOH o 2 mg/ml Zymolyase-20T (added after autoclaving). o Transfer to an Eppendorf tube and incubate at 37C for 1hour. 4. Pellet the cells in an Eppendorf centrifuge for 30 seconds. Resuspend in 300 l TE. 5. Add 35 l 10% SDS, mix and incubate at 65C for 5 minutes. 6. Add 100 l 5M potassium acetate, mix and leave on ice for 30 minutes. 7. Spin down at 4C for 10 minutes. 8. Add 50 l of supernatant to 100 l NaI solution (Geneclean Kit, Stratech Scientific Ltd.) with 5 l glassmilk (Geneclean Kit, Stratech Scientific Ltd.). 9. Incubate for 5 minutes at room temperature. 10. Spin for 5 seconds (maximum) at room temperature, discard the supernatant and wash the pellet three times with 400 l of ice-cold NEW wash (Geneclean Kit, Stratech Scientific Ltd.). 11. Elute DNA twice with 10 l of TE at 55C for 3 minutes each time. 12. Spin out the glass milk and keep the supernatant. 13. Transform 5 l of the supernatant into 100 l of competent E.coli JA226 cells. The use of the Geneclean or Qiagen kits improves the transformation frequency by at least 10 fold; also it is very important to use a recBC E.coli strain such as JA226 when using 2 m or non-ars containing plasmids. For ars1 based plasmids recA strains like E. coli DH5 can be used. Procedure B A second method is also used; this has been less well tried in this lab but is quicker and reportedly gives good results, as follows: 1. Grow small cultures (at least 1.4 ml) with selection. 2. Collect the cells by a 5 second spin in a microfuge. 3. Decant away the supernatant and briefly vortex the tube to resuspend the pellet in the residual medium. 4. Add 0.2 ml of: o 2 % Triton X-100 o 1 % SDS o 100mM NaCl o 10mM Tris HCl (pH 8.0)

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o 1mM Na2EDTA? 5. Add 0.2 ml phenol:chloroform:IAA and 0.3 g acid washed glass beads. 6. Vortex for 2 minutes and then microfuge for 5 minutes. 7. Take upper aqueous layer to a fresh Eppendorf and extract with 200l phenol:chloroform:IAA. 8. Precipitate the DNA , wash with 70% EtOH, dry and resuspend in 10l TE. 9. Use 1-5 l for transformations of competent E.coli or yeast .

Note: This method also works well for isolating genomic DNA.

2.8 Multicopy overexpression of genes under the control of the nmt promoter
The standard Thiamine concentration for repression of gene expression from the nmt-1,-41 and -81 promoters is 15M (200l per 400ml medium of a 10mg/ml stock solution stored in the dark at 4C), although 2M Thiamine is usually sufficient for repression of the promoter in most cases, and may aid induction. Certain "toxic" gene products require extra suppression and growth can be aided by increasing the concentration up to 60M especially when using nmt1 which is the strongest and leakiest of these promoters. Despite the above, it is not really possible to partially induce the promoter in a population by using low thiamine concentrations. If overexpression of a gene product is toxic to the cells, they will quickly be selected for containing recombinant plasmids that are less toxic. These cells may rapidly overgrow the other cells. Addition of Thiamine during the experiment may help to keep concentrations topped up! A Thiamine concentration of 2nM in minimal medium plates is sufficient for cells overexpressing prep3x-nmt1-rum1, to grow to a colony size of 0.1-1mm in three days at 32C, before running out of Thiamine. Such colonies consist of cells showing the rum1 overexpression phenotype (which ran out of Thiamine before running out of nutrition - at the border of the colony) and of starved wild-type cells (which ran out of nutrition before running out of Thiamine - in the centre of the colony). Although the rum1 overexpression phenotype is lethal the strains can be recovered very easily by transferring the starved cells to +Thiamine medium. This method has been used in a screen designed to isolate cDNAs whose products alter cell morphology when overexpressed. It has the advantage that no replica plating from +Thiamine to -Thiamine plates is required. The minimal Thiamine concentration mentioned above might have to be increased or further decreased to give similar results when overexpressing other genes and/or when using another promoter. Promoter strength (and leakiness): nmt1 > nmt41 > nmt81 If a strain is used for a screen containing ectopic expression of a gene from the nmt promoter, mutations frequently affect the activity of the nmt promoter. These should be screened out in a secondary screen, e.g. by Northern Analysis.