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A modified medium for efficient electrotransformation of E. coli
TECHNICAL
M e
~j~
bp
bp
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7 8 9 10 laW,tam I. Ethidium bromide-stained agarose gel demonstrating the applicability of automatic PCR separation to pool screening. Lane 3, homoz3,gous normal genomic DNA (N). A serial dilution of genomic DNA from a CFTR R1162X heterozygous individual with increasing amounts of normal genomic DNA (N) was subjected to the CFTR Rl162X-specific PCR assay (lanes 4-10). The ratios of mutant to normal DNA are indicated above each lane. Lanes 3-10 all show the 457 bp band of the pre-PCR. The 163 bp mutation-specific fragments in lanes 4-10 indicate presence of the CFTRR1162X mutation in the respective samples. As a control, the same reaction without any DNA template is shown in lane 2 (B). Lane 1 contains 0.5 Izg HaeIII-digested pBR322 (M).
TIPS
We have successfully applied this principle to four different point mutations. The system is capable of detecting one mutant allele in the presence of up to 1200 wild-type alleles (Fig. 1). In these experiments, PCR reactions were performed with 50 mM KCI, 10 mM Tds-HCI, 0.1% Triton X-100, 1.5 mM MgCI2, 0.1 mM dNTPs and 0.25 lag genomic DNA. Primer concentrations were as follows: 0.4 IzM oligonucleotide 1 (5'-GCCCGACAAATAACCAAGTGACAAATAGCAAGTGTTGC-3'); 0.008 luuM oligonucleotide 2 (5'-TCTGCTAACACATrGCT TCAGGCTACTGGGATrCA-3'); 0.4 IzM oligonucleotide 3 (5'-TCAATGAACTrAAAGACrAA-3'). Initial denaturation was at 95°C, 3 min, and was followed by a 'hot start'. Thermal cycling was carried out for 15 cycles as follows: 94°C, 30 s; 65°C, 30 s; 72°C, 40 s. This was followed by 25 cycles as follows: 94°C, 30 s; 45°C, 30 s; 72°C, 30 s with a time increment of 3 s/cycle for the latter temperature. Taq DNA polymerase lacking 3'-5' exonuclease activity was used (Stratagene). Apart from screening pools of genomic DNAs, this method might also be useful for library screening. REFERENCE
1 Ruzicka, V., Miirz, W., Russ, A. and Gross, W. (1992) J. Lipid Res. 33, 1563--1567
Contributed by Andreas Orou, Birga Fecbne~, Hans-Jfo~,en Menzel and Gerd Utema~nn, Institutffir Mediziniscbe Biologie und Humangenetik der UniversitditInnsbruck, SchOpfitrafle 41, A-6020 Innsbruck, Austria.
A modified medimn for efficient electrotransformation of E colt Transformation of E. coli is an important technique in molecular biology. Efficiency of transformation is of concern when only a small amount of DNA is available, or when constructing No. transformantsa (× 101° wg-1 of pUC18) a representative cDNA library of rarely expressed genes. The application of electroporation has substantially improved transStrain 10% glycerol GYT medium formation efficiencies, and an efficiency of 109 transformants per l~g of plasmid DNA has been reported1, z. We find that ff JM109 0.20 + 0.08 2.22 + 0.12 tryptone and yeast extract are included in the electrotransHB101 0.15 + 0.04 1.03 + 0.10 formation medium, we can achieve 1010 transformants per l~g of Sureb 0.29 +-0i15 1.32-+ 0.09 plasmid: DHI 0,54 + 0,19 2.45 + 0.31 Bacteria are made competent as follows. 100 ml of LB medium NM538 0.10 + 0.03 0.99 + 0.16 is inoculated with 1 ml of an overnight culture of bacteria RR1 0,30 + 0.07 2.20 + 0.31 and incubated at 37°C with vigorous shaking. When the OD600 of the culture reaches 0.6, it is chilled on ice for 30 min. Cells are aValues are means -+SEM of three independent harvested at 4000 g for 15 min at 4°C, washed twice with 50 ml measurements. of ice-cold 10% glycerol, and resuspended to a final volume of bSure is a strain developed by Stratagene. 0.2 ml in ice-cold GYT (10% glycerol, 0.125% yeast extract and 0.25% tryptone). Electrotransformation is performed using a Gene Pulser (BioRad) in a prechilled 0.2 ml cuvette, under conditions suggested by the manufacturer (i.e. 25 p,F, 200 1"1, 2.5 kV). 0.5 ng of pUC18 is used per transformation. After electrotransformation, 1 ml of SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 10 mM MgSO4,10 mM MgCl2) is added immediately. Ceils are transferred to a polypropylene tube (17 mm × 100 ram) and incubated at 37°C for I h to allow expression of the phenotype conferred by the selected locus. Cells are then seeded on selective agar plates and incubated at 37°C overnight. Inclusion of yeast extract and tryptone in the electrotransformation medium consistently increased the number of transformants 4-10-fold over that obtained using standard medium, 10% glycerol. This enhancement does not appear to be restricted to any specific E. colistrain: all six strains tested, which are commonly used in molecular biololgy, showed similar responses. Media containing higher concentrations of both tryptone and yeast extract have been tried, but caused sparks and arcing, while media with lower concentrations gave less promising yields. Survival of cells in the electrotransformed cultures has been investigated. Cells electrotransformed in GYT medium had essentially the same survival rate as those transformed in standard medium. It seems likely that the nutrients added confer more efficient phenotypic expression on the electroporated cells, thus enhancing the number of transformants recovered. Competent cells prepared in this modified medium can be stored in aliquots at -80°C without impairing cell survival or transformation efficiency; however, repeated freezing and thawing of the cells should be avoided.
T~mL~1. Transfo,~mation efficiency orE. co//strains usin8 various electrotransformation media
TIG APRIL 1995 VOL. 11 NO. 4 ,~ 109"~ Elsevier Science Ltd (1168 - 052"5/95/$09.50
128
TECHNICAL
TIPS
REFERENCES
1 Lee, S.Y. and Chang, H.N. (1994) BioTecbniques 16, 206-208 2 Siguret, V. et al. (1994) BioTechniques 16, 422-426
Contributed by Wai Lin Tung and K/ng-C Chow, Depm¢ment of Biochemistry, The Hong Kong Univemitl, of Science and Technology, Clear Water Ba3; Ko'~loon, Hong Kong.
Rapid fluorescence in situ detection of heterologous expression in E. coil and counterstaining with diamidinophenylindol We describe a rapid method for the identification of bacteria expressing heterologous genes on microscope slides. Heterologous mRNA is detected directly by antisense fluorescence in situ hybridization (AS-FISH). DAFI, a fluorescent dye normally used to identify human chromosomes or interphase nuclei, is used for counterstaining of E. coli cells. This allows simultaneous identification of induced bacteria and a morphological analysis of the bacteria. Furthermore, relocation of interesting slide regions after DAPI staining under fluorescent microscopy conditions is straightforward. Alternative methods for the identification of E. coli cells expressing heterologous genes include Northern blot analysis, RT-PCR and radiolabelled in situ hybridization in combination with light microscopy for morphological examination of the cells. Our method is more rapid than these, produces complete results within only three days and avoids radioactivity.
Procedure 1 E. coli cells (SM1, wt: BL 26) were grown up to naid-logarithmic phase in LB medium and were fLxed in paraformaldehyde according to Ref. 1. Abom 10 p,l of these cells were transferred to slides coated with 3-aminopropyttriethoxysilane and fixed by air drying overnight. 2 Bacteria expressing the human PMP22 gene 2-4 were detected by AS-FISH (Fig. la). This technique involves slight modifications of the standard FISH protocol (e.g. Ref. 3). The main modifications were: (i) the use of HPLC-purified oligonucleotides biotinylated during synthesis as sense and antisense probes (in our case 42mers specific for a highly cor~served PMP22 region); (ii) a hybridization buffer containir~ . . . . /_1 total yeast RNA and 0.1 mg/ml yeast transfer RNA, (iii) no & of the target tissue, (iv) a hybridization temperature of 47°(2, ." use of RNAse free solutions. Detection of biotinylated probe formed using a FITC-avidin system. 3 E. coli cells were counterstained with a DAPI (diamidinopl: solution for 8 min (5 I~g DAPI/100 ml 4 × SSC/0.2% Tween). rinsed in double-distilled water about four times, for a few sec For microscopy some antifade solution and a coverslip were airdried slides. 4 Evaluation of DAPI and FITC signals was performed at 340~ 450-490nm wavelength respectively with a fluorescence and a 1000X objective with oil immersion. The result of DA (Fig. lb), shows even morphological details of the blue-color~ REFERENCES
1 Amann, R.I. et al. (1990) Appl. Environ. Microbiol. 56, 1919-1925 2 Patel, P.I. and Lupski, J.R. (1994) Trends Genet. 10, 128-132 3 Kraus, C. et al. (1994) Genomics 23, 272-274
Contributed by Thomas Liehr, Heike Altenberger and Bernd Rautenstrauss, Institute for Human Genetics, Schwabachanlage 10, D-91054 Erlangen, Germany.
(Photographs were taken by a Leitz Orthomat, on Fujichrome 1600 film.) (a) AS-FISH to E. coli expressing human PMP22. Two groups of cells are visible: one group with strong expression, showing bright green FITC-staining, and a second showing weak or no expression in this stage of cell cycle. (b) DAPI staining of these cells. (c) Negative control, using the complementary (sense) probe to the PMP22probe used in part (a). No signal is visible in the cells.
Authors' corroL~on DNA extraction from urea-preserved blood or blood clots for use in PCR by Annette Gelhaus, Britta Urban and Claude Pirmez Trends in Genetics 11, 41 The protocol in this technical tip contained an error. Step 5 should be omitted, and the protocol should proceed direcdy from step 4 to step 6. TIG APPalL 1995 VOL. 11 NO. 4 © 1995 Elsevier Science Ltd 0168 - 9525/95/$09.50
129
M e
~j~
bp
bp
--~,,
7 8 9 10 laW,tam I. Ethidium bromide-stained agarose gel demonstrating the applicability of automatic PCR separation to pool screening. Lane 3, homoz3,gous normal genomic DNA (N). A serial dilution of genomic DNA from a CFTR R1162X heterozygous individual with increasing amounts of normal genomic DNA (N) was subjected to the CFTR Rl162X-specific PCR assay (lanes 4-10). The ratios of mutant to normal DNA are indicated above each lane. Lanes 3-10 all show the 457 bp band of the pre-PCR. The 163 bp mutation-specific fragments in lanes 4-10 indicate presence of the CFTRR1162X mutation in the respective samples. As a control, the same reaction without any DNA template is shown in lane 2 (B). Lane 1 contains 0.5 Izg HaeIII-digested pBR322 (M).
TIPS
We have successfully applied this principle to four different point mutations. The system is capable of detecting one mutant allele in the presence of up to 1200 wild-type alleles (Fig. 1). In these experiments, PCR reactions were performed with 50 mM KCI, 10 mM Tds-HCI, 0.1% Triton X-100, 1.5 mM MgCI2, 0.1 mM dNTPs and 0.25 lag genomic DNA. Primer concentrations were as follows: 0.4 IzM oligonucleotide 1 (5'-GCCCGACAAATAACCAAGTGACAAATAGCAAGTGTTGC-3'); 0.008 luuM oligonucleotide 2 (5'-TCTGCTAACACATrGCT TCAGGCTACTGGGATrCA-3'); 0.4 IzM oligonucleotide 3 (5'-TCAATGAACTrAAAGACrAA-3'). Initial denaturation was at 95°C, 3 min, and was followed by a 'hot start'. Thermal cycling was carried out for 15 cycles as follows: 94°C, 30 s; 65°C, 30 s; 72°C, 40 s. This was followed by 25 cycles as follows: 94°C, 30 s; 45°C, 30 s; 72°C, 30 s with a time increment of 3 s/cycle for the latter temperature. Taq DNA polymerase lacking 3'-5' exonuclease activity was used (Stratagene). Apart from screening pools of genomic DNAs, this method might also be useful for library screening. REFERENCE
1 Ruzicka, V., Miirz, W., Russ, A. and Gross, W. (1992) J. Lipid Res. 33, 1563--1567
Contributed by Andreas Orou, Birga Fecbne~, Hans-Jfo~,en Menzel and Gerd Utema~nn, Institutffir Mediziniscbe Biologie und Humangenetik der UniversitditInnsbruck, SchOpfitrafle 41, A-6020 Innsbruck, Austria.
A modified medimn for efficient electrotransformation of E colt Transformation of E. coli is an important technique in molecular biology. Efficiency of transformation is of concern when only a small amount of DNA is available, or when constructing No. transformantsa (× 101° wg-1 of pUC18) a representative cDNA library of rarely expressed genes. The application of electroporation has substantially improved transStrain 10% glycerol GYT medium formation efficiencies, and an efficiency of 109 transformants per l~g of plasmid DNA has been reported1, z. We find that ff JM109 0.20 + 0.08 2.22 + 0.12 tryptone and yeast extract are included in the electrotransHB101 0.15 + 0.04 1.03 + 0.10 formation medium, we can achieve 1010 transformants per l~g of Sureb 0.29 +-0i15 1.32-+ 0.09 plasmid: DHI 0,54 + 0,19 2.45 + 0.31 Bacteria are made competent as follows. 100 ml of LB medium NM538 0.10 + 0.03 0.99 + 0.16 is inoculated with 1 ml of an overnight culture of bacteria RR1 0,30 + 0.07 2.20 + 0.31 and incubated at 37°C with vigorous shaking. When the OD600 of the culture reaches 0.6, it is chilled on ice for 30 min. Cells are aValues are means -+SEM of three independent harvested at 4000 g for 15 min at 4°C, washed twice with 50 ml measurements. of ice-cold 10% glycerol, and resuspended to a final volume of bSure is a strain developed by Stratagene. 0.2 ml in ice-cold GYT (10% glycerol, 0.125% yeast extract and 0.25% tryptone). Electrotransformation is performed using a Gene Pulser (BioRad) in a prechilled 0.2 ml cuvette, under conditions suggested by the manufacturer (i.e. 25 p,F, 200 1"1, 2.5 kV). 0.5 ng of pUC18 is used per transformation. After electrotransformation, 1 ml of SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 10 mM MgSO4,10 mM MgCl2) is added immediately. Ceils are transferred to a polypropylene tube (17 mm × 100 ram) and incubated at 37°C for I h to allow expression of the phenotype conferred by the selected locus. Cells are then seeded on selective agar plates and incubated at 37°C overnight. Inclusion of yeast extract and tryptone in the electrotransformation medium consistently increased the number of transformants 4-10-fold over that obtained using standard medium, 10% glycerol. This enhancement does not appear to be restricted to any specific E. colistrain: all six strains tested, which are commonly used in molecular biololgy, showed similar responses. Media containing higher concentrations of both tryptone and yeast extract have been tried, but caused sparks and arcing, while media with lower concentrations gave less promising yields. Survival of cells in the electrotransformed cultures has been investigated. Cells electrotransformed in GYT medium had essentially the same survival rate as those transformed in standard medium. It seems likely that the nutrients added confer more efficient phenotypic expression on the electroporated cells, thus enhancing the number of transformants recovered. Competent cells prepared in this modified medium can be stored in aliquots at -80°C without impairing cell survival or transformation efficiency; however, repeated freezing and thawing of the cells should be avoided.
T~mL~1. Transfo,~mation efficiency orE. co//strains usin8 various electrotransformation media
TIG APRIL 1995 VOL. 11 NO. 4 ,~ 109"~ Elsevier Science Ltd (1168 - 052"5/95/$09.50
128
TECHNICAL
TIPS
REFERENCES
1 Lee, S.Y. and Chang, H.N. (1994) BioTecbniques 16, 206-208 2 Siguret, V. et al. (1994) BioTechniques 16, 422-426
Contributed by Wai Lin Tung and K/ng-C Chow, Depm¢ment of Biochemistry, The Hong Kong Univemitl, of Science and Technology, Clear Water Ba3; Ko'~loon, Hong Kong.
Rapid fluorescence in situ detection of heterologous expression in E. coil and counterstaining with diamidinophenylindol We describe a rapid method for the identification of bacteria expressing heterologous genes on microscope slides. Heterologous mRNA is detected directly by antisense fluorescence in situ hybridization (AS-FISH). DAFI, a fluorescent dye normally used to identify human chromosomes or interphase nuclei, is used for counterstaining of E. coli cells. This allows simultaneous identification of induced bacteria and a morphological analysis of the bacteria. Furthermore, relocation of interesting slide regions after DAPI staining under fluorescent microscopy conditions is straightforward. Alternative methods for the identification of E. coli cells expressing heterologous genes include Northern blot analysis, RT-PCR and radiolabelled in situ hybridization in combination with light microscopy for morphological examination of the cells. Our method is more rapid than these, produces complete results within only three days and avoids radioactivity.
Procedure 1 E. coli cells (SM1, wt: BL 26) were grown up to naid-logarithmic phase in LB medium and were fLxed in paraformaldehyde according to Ref. 1. Abom 10 p,l of these cells were transferred to slides coated with 3-aminopropyttriethoxysilane and fixed by air drying overnight. 2 Bacteria expressing the human PMP22 gene 2-4 were detected by AS-FISH (Fig. la). This technique involves slight modifications of the standard FISH protocol (e.g. Ref. 3). The main modifications were: (i) the use of HPLC-purified oligonucleotides biotinylated during synthesis as sense and antisense probes (in our case 42mers specific for a highly cor~served PMP22 region); (ii) a hybridization buffer containir~ . . . . /_1 total yeast RNA and 0.1 mg/ml yeast transfer RNA, (iii) no & of the target tissue, (iv) a hybridization temperature of 47°(2, ." use of RNAse free solutions. Detection of biotinylated probe formed using a FITC-avidin system. 3 E. coli cells were counterstained with a DAPI (diamidinopl: solution for 8 min (5 I~g DAPI/100 ml 4 × SSC/0.2% Tween). rinsed in double-distilled water about four times, for a few sec For microscopy some antifade solution and a coverslip were airdried slides. 4 Evaluation of DAPI and FITC signals was performed at 340~ 450-490nm wavelength respectively with a fluorescence and a 1000X objective with oil immersion. The result of DA (Fig. lb), shows even morphological details of the blue-color~ REFERENCES
1 Amann, R.I. et al. (1990) Appl. Environ. Microbiol. 56, 1919-1925 2 Patel, P.I. and Lupski, J.R. (1994) Trends Genet. 10, 128-132 3 Kraus, C. et al. (1994) Genomics 23, 272-274
Contributed by Thomas Liehr, Heike Altenberger and Bernd Rautenstrauss, Institute for Human Genetics, Schwabachanlage 10, D-91054 Erlangen, Germany.
(Photographs were taken by a Leitz Orthomat, on Fujichrome 1600 film.) (a) AS-FISH to E. coli expressing human PMP22. Two groups of cells are visible: one group with strong expression, showing bright green FITC-staining, and a second showing weak or no expression in this stage of cell cycle. (b) DAPI staining of these cells. (c) Negative control, using the complementary (sense) probe to the PMP22probe used in part (a). No signal is visible in the cells.
Authors' corroL~on DNA extraction from urea-preserved blood or blood clots for use in PCR by Annette Gelhaus, Britta Urban and Claude Pirmez Trends in Genetics 11, 41 The protocol in this technical tip contained an error. Step 5 should be omitted, and the protocol should proceed direcdy from step 4 to step 6. TIG APPalL 1995 VOL. 11 NO. 4 © 1995 Elsevier Science Ltd 0168 - 9525/95/$09.50
129