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Utilizing Bioinformatics

Utilizing Bioinformatics. Cloning and expression in E. coli. Useful Websites for Gene Cloning Using Bioinformatics. Portal sites Ecogene : http://ecogene.org/ Bioinformatics organization: http://www.bioinformatics.org/ Expasy : http://www.expasy.org/ NCBI: http://www.ncbi.nlm.nih.gov/

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Utilizing Bioinformatics

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  1. Utilizing Bioinformatics Cloning and expression in E. coli

  2. Useful Websites for Gene Cloning Using Bioinformatics Portal sites Ecogene: http://ecogene.org/ Bioinformatics organization: http://www.bioinformatics.org/ Expasy: http://www.expasy.org/ NCBI: http://www.ncbi.nlm.nih.gov/ Protein data bank: http://www.rcsb.org/pdb/home/home.do Tools Reverse complement: http://www.bioinformatics.org/sms/rev_comp.html ORF (open reading frame) finder: http://www.ncbi.nlm.nih.gov/projects/gorf/ DNA translation: http://web.expasy.org/translate/

  3. pMAL-TEV-XhoI TEV site: ENLYQS/G → ENLYQ + S/G TEV site

  4. PCR condition Mixture of PCR reaction -Forward primer: 5 ml of 10 pmole -Reverse primer: 5 ml of 10 pmole -Template: 1 ml of DNA -dNTP (2.5 mM each): 5 ml -10X reaction buffer: 5 ml -Pfu DNA polymerase: 1 ml -DDW: 28 ml Total: 50 ml PCR running condition -Initial denaturation: 94°C, 3 min -Denaturation: 94°C, 45 sec -Annealing: 55°C, 45 sec -Extension: 72°C, 1 min (1min/kb) -Final extension: 72°C, 5 min 25- 30 cycles

  5. Restriction enzyme digestion for PCR product *Digest both PCR product and plasmid with the same restriction enzymes Purification of PCR product -Use a PCR purification kit -According to the instruction of vender *Elute with DDW of40 ml Mixture of digestion -DNA: 40 ml (purified PCR product) -NdeI: 2.5 ml -XhoI: 2.5 ml -10X NEB buffer(#2): 5 ml Total: 50 ml Digestion condition -37°C, 3 hr Purification -According to the instruction of vender *Elute with DDW of30 ml

  6. Restriction enzyme digestion for plasmid *Digest both PCR product and plasmid with the same restriction enzymes Mixture of digestion -DNA: 40 ml (purified plasmid) -NdeI: 2.5 ml -XhoI: 2.5 ml -10X NEB buffer(#2): 5 ml Total: 50 ml Digestion condition -37°C, 3 hr CIP treatment -DNA & RE mixture: 50 ml -CIP: 2 ml (37°C, 1 hr) Purification -According to the instruction of vender *Elute with DDW of30 ml

  7. Ligation condition *Run agarose gel electrophoresis to verify DNA purification before ligation ! Mixture of ligation -Insert: 3 ml (digested PCR product) -Plasmid: 2 ml (digested pMAL-TEV-XhoI) -2X ligation mixture (solution I): 5 ml Total: 10 ml Ligation condition -16°C, over 6 hr Transformation -Competent cell (E. coli MC1061): 200 ml -Ligation mixture: 10 ml -Cold shock: 0°C (on ice), over 10 min -Heat shock: 42°C, 90 sec -Plating on LBA plasmid

  8. Confirmation of cloning via colony PCR Mixture of colony PCR reaction -Forward primer: 1 ml of 10 pmole (pMAL-F) -Reverse primer: 1 ml of 10 pmole (pMAL-R) -Template: 1 colony -dNTP (2.5 mM each): 2 ml -10X reaction buffer: 2 ml -TaqDNA polymerase: 1 ml -DDW: 15 ml Total: 20 ml PCR running condition -Initial denaturation: 94°C, 3 min -Denaturation: 94°C, 45 sec -Annealing: 55°C, 45 sec -Extension: 72°C, 1 min (1min/kb) -Final extension: 72°C, 5 min 25- 30 cycles *Run agarose gel electrophoresis after PCR to confirm the insertion of target DNA into the plasmid!

  9. Confirmation of correct cloning via RE digestion *Extract plasmid from the positive colonies. (plasmid preparation) Mixture of digestion -DNA: 8 ml (purified plasmid) -NdeI: 0.5 ml -XhoI: 0.5 ml -10X NEB buffer(#2): 1 ml Total: 10 ml (37°C, over 1 hr) *Run agarose gel electrophoresis after RE digestion. *There should be two bands on the agarose gel, which represent plasmid and insert DNA.

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