Digesting Plasmid DNA with Restriction Endonuclease s under the Condition of Microwave-heating

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1 ISSN CN / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ * min DNA 2 min Q78 Digesting Plasmid DNA with Restriction Endonuclease s under the Condition of Microwave-heating 1 MU Hui-Min 2 JIANG Hua 1 WANG Yan-Li 1 SUN Guo-Chang 1 * 1 Institute of Plant Protection and Microbiology Zhejiang Academy of Agricultural Sciences Hangzhou 2 Institute of Plant Protection Nanjing Agricultural University Nanjing China China Abstract During molecular biological experiments restriction endonuclease-digestion was used frequently but the digestion reaction normally needs to incubate the mixture in water bath for a long time The microwave was thought to be able to catalyze restriction endonuclease-digestion and save the incubation time In this study we designed a series of experiments to verify whether the microwave really could catalyze the reaction The results showed that DNA degradation and enzyme inactivation didn t happen when the restriction endonuclease- digestion reaction system was treated by microwave at high temperature for 2 min For plasmid digestion with one or two restriction endonucleases treatment by microwave for short time 2 min could alternate water bath to complete digestion reaction For some double digestion systems microwave digestion is not complete and can t substitute for water bath The digestion time had to control strictly when the microwave was used to catalyze the digestion Key words enzyme digestion reaction microwave oven microwave No Y No No * 2 ~ 4 h 5 37 * Tel sungc@ zaas org Received September Accepted January Supported by Natural Science Foundation of Zhejiang Province No Y National Natural Science Foundation of China No No Corresponding author Tel sungc@ zaas org

2 Guy11 Guy11 10 μl 1% 0 5 TBE 39 Mb 6 EB 389 Mb 7 pcambia ~ μl 1 5 μl 2 25 kb TaKaRa Fermentas μg EcoR Ⅴ 15 U / μl / BstX Ⅰ 10 U / μl Galanz WD850 TaKaRa 0 3 μl 10 Buffer 1 μl H 2 O 7 2 μl D8523TL-2H 220 V ~ 50 Hz W W 2 min 10 s 2 min 850 W 100% 81% 58% 36% 18% 1 2 BstXⅠ 42 EcoRⅠ DNA EDTA Guy11 10 mmol / L 10 Loading Buffer 2 μl DNA 11 ~ 12 5 μl min 10 Loading Buffer 1% 0 5 TBE EB μl 2 μl 3 μg 10 Buffer 1 μl Sal Ⅰ / EcoR Ⅴ TaKaRa 15 U / μl Fermentas 10 U / μl 0 3 μl H 2 O 6 7 μl 30 s TBE EB min 1 min 30 s 2 min 3 min 5 min 2 μl 3 Table 1 Plasmid double digestion system TaKaRa 15 U / μl μg 37 2 h 37 2 h EDTA 10 mmol / L 10 Loading Buffer 3 2 min 10 min 30 min 1 h 2 h 3 h 4 h 1% 0 5 TBE EB 4 10 μl Table 1 2 min 10 s 2 min min 10 min 2 h 3 h EDTA 10 mmol / L 10 Loading Buffer 2 μl 1% Fermentas 10 U / μl Plasmid 1 5 μl Plasmid 1 5 μl Plasmid 1 5 μl Plasmid 1 5 μ HindⅢ 0 3 μl EcoRⅠ 0 3 μl HindⅢ 0 6 μl EcoRⅠ 0 3 μ BamHⅠ 0 3 μl PstⅠ 0 3 μl BamHⅠ 0 3 μl PstⅠ 0 3 μ 10 Buffer K 1 0 μl 10 Buffer H 1 0 μl 10 Buffer BamHⅠ 1 0 μl 10 Buffer O 1 0 μ H 2 O 6 9 μl H 2 O 6 9 μl H 2 O 6 6 μl H 2 O 6 9 μ * Plasmid 1 5 μl 2 25 μg Ⅴ Fermentas 2 min 3 min 5 min 5 min Fig 2 10 min Guy11 2 min 3 min 5 min 10 min 2 min 3 min 5 min 10 min Fig 1 2 min DNA Fig 3 min 1 min 1 5 min 2 min 3 min EcoR 2 min h 2 h 3 h 4 h 37 2 min 10 min 30 min 37 1 h 2 h 3 h 4 h

3 Fig 1 The integrity of plasmid and genome after microwave treatment Restriction endonuclease-digestion product electrophoresed on 1% agarose gel in 0 5 TBE buffer run at 160 V for 35 min and then stained with ethidium bromide The gel image was recorded by using Gel Image System A Plasmid B Rice blast Guy11 genome C Rice genome D High for 10 min M GenRuLer TM 1 kb DNA ladder 1 High for 2 min 2 High for 3 min 3 High for 5 min 4 Control Fig 2 The activity of restriction endonuclease after microwave treatment Restriction endonuclease digestion product electrophoresed on 1% agarose gel in 0 5 TBE buffer run at 160 V for 35 min and then stained with ethidium bromide The gel image was recorded by using Gel Image System A High for 0 5 min B High for 1 min C High for 1 5 min D High for 2 min E High for 3 min F High for 5 min G Control M GenRuLer TM 1 kb DNA ladder 1 EcoRⅠ TaKaRa 2 EcoRⅤ TaKaRa 3 EcoRⅤ Fermentas 4 SalⅠ Fermentas Fig 3 Plasmid restriction endonuclease digestion Restriction endonuclease digestion product electrophoresed on 1% agarose gel in 0 5 TBE buffer run at 160 V for 35 min and then stained with ethidium bromide The gel image was recorded by using Gel Image System A EcoRⅤ B BstXⅠ M GenRuLer TM 1 kb DNA ladder 1 High continuously 2 High interval 3 Medhigh continuously 4 Med-high interval 5 Med continuously 6 Med interval 7 Defrost continuously 8 Defrost interval 9 Low continuously 10 Low interval min min min h h h h

4 4-383 PstⅠ / EcoR Ⅰ TaKaRa Hind Ⅲ / Bam H Ⅰ TaKaRa Pst Ⅰ / EcoR Ⅰ 37 2 min 10 min 37 2 h 3 h Fermentas Fig min 10 min 2 h 3 h 2 min HindⅢ / Bam H Ⅰ Fermentas Fig 4 Plasmid double digestion Restriction endonuclease digestion product electrophoresed on 1% agarose gel in 0 5 TBE buffer run at 160 V for 35 min and then stained with ethidium bromide The gel image was recorded by using Gel Image System A PstⅠ / EcoRⅠ TaKaRa B Hind Ⅲ / BamH Ⅰ TaKaRa C Pst Ⅰ / EcoR Ⅰ Fermentas D BamH Ⅰ / Hind Ⅲ Fermentas M GenRuLer TM 1 kb DNA ladder 1 High continuously 2 High interval 3 Low continuously 4 Low interval min min h h 3 5 min min 3 14 min 5 min 15 2 min 5 2 min - 2 min DNA DNA 2 min Guy11 TaKaRa 10 Buffer Fermentas 10 Buffer

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