Transcript Cloning and expression of slyD from E. coli

Utilizing Bioinformatics
Cloning and expression in E. coli
Useful Websites for Gene Cloning Using Bioinformatics
Portal sites
1. Ecogene: http://ecogene.org/
2. Bioinformatics organization: http://www.bioinformatics.org/
3. Expasy: http://www.expasy.org/
4. NCBI: http://www.ncbi.nlm.nih.gov/
5. Protein data bank: http://www.rcsb.org/pdb/home/home.do
Tools
1. Reverse complement:
http://www.bioinformatics.org/sms/rev_comp.html
2. ORF (open reading frame) finder:
http://www.ncbi.nlm.nih.gov/projects/gorf/
3. DNA translation: http://web.expasy.org/translate/
pMAL-TEV-XhoI
lacI
pBR322 origin
tac promoter
pMAL-TEV-XhoI
6635 bp
malE
TEV protease site
Nde I (2662)
EcoRI (267 6)
BamHI (2682)
Amp
HindIII (27 08)
TEV site: ENLYQS/G → ENLYQ + S/G
Xho I (27 1 4)
rrnB T1T2
TEV site
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
25- 30 cycles
-Annealing: 55°C, 45 sec
-Extension: 72°C, 1 min (1min/kb)
-Final extension: 72°C, 5 min
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 of 40 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 of 30 ml
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 of 30 ml
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
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
-Taq DNA polymerase: 1 ml
-DDW: 15 ml
Total: 20 ml
PCR running condition
-Initial denaturation: 94°C, 3 min
-Denaturation: 94°C, 45 sec
25- 30 cycles
-Annealing: 55°C, 45 sec
-Extension: 72°C, 1 min (1min/kb)
-Final extension: 72°C, 5 min
*Run agarose gel electrophoresis after PCR to confirm the insertion of
target DNA into the plasmid!
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.