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Bioinformatics Tools and Techniques for Cloning and Expression in E. coli

This guide provides an overview of bioinformatics resources and practical protocols for cloning and expressing genes in E. coli. Included are essential websites such as Ecogene, NCBI, and Expasy, offering tools for gene analysis and protein structure. The cloning process steps are detailed, from PCR setup, restriction enzyme digestion, purification of PCR products, to ligation and transformation into competent cells. Key experimental conditions and troubleshooting tips are included to facilitate successful gene cloning and expression studies.

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Bioinformatics Tools and Techniques for Cloning and Expression in E. coli

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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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