Team:HokkaidoU Japan/Notebook/overall protocols
From 2012.igem.org
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(Mini-prep) |
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==Transformation== | ==Transformation== | ||
+ | <p> | ||
#Add 1~2 ul of DNA to 50 ul of thawed competent cells on ice. | #Add 1~2 ul of DNA to 50 ul of thawed competent cells on ice. | ||
#Incubate on ice for 30 min. | #Incubate on ice for 30 min. | ||
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#Add 900 ul of LB to 100 ul of the culture and plate 300 ul of it onto second dish and spread. | #Add 900 ul of LB to 100 ul of the culture and plate 300 ul of it onto second dish and spread. | ||
#Incubate the plates at 37C for 16~20 hours. | #Incubate the plates at 37C for 16~20 hours. | ||
+ | </p> | ||
+ | |||
+ | ==Mini-prep== | ||
+ | <p> | ||
+ | We are using mini-prep kit of Nippon genetics: FastGene Plasmid mini kit. | ||
+ | This kit contains these reagents and wares: mP1, mP2, mP3, mP4, mP5, mP6, column and collection tube. | ||
+ | #Centrifuge 1~5 ml of culture at over 10,000 rpm for 2 min. | ||
+ | #Remove the supernatant. | ||
+ | #Add 200ul of mP1 then voltexing. | ||
+ | #Add 200ul of mP2 and invert the tube then leave for 2 min at room temperature. | ||
+ | #Add 200ul of mP3 then invert the tube. | ||
+ | #Centrifuge at 1,3000 rpm for 8 min. | ||
+ | #Centrifuge 1,3000 rpm for 1 min to load supernatant from column to collection tube. | ||
+ | #Remove filtrate and add 400 ul of mP4 then centrifuge 13,000 rpm for 1 min. | ||
+ | #Remove filtrate and add 600 ul of mP5 then centrifuge 13,000 rpm for 1 min. | ||
+ | #Remove filtrate and centrifuge 13,000 rpm for 2 min. | ||
+ | #Set column into 1.5 ml centrifuge tube. | ||
+ | #Add 50 ul of mP6. | ||
+ | #Centrifuge at 13,000 rpm for 2 min. | ||
+ | </p> | ||
Revision as of 10:22, 10 August 2012
Transformation
- Add 1~2 ul of DNA to 50 ul of thawed competent cells on ice.
- Incubate on ice for 30 min.
- Add 600 ul of LB.
- Incubate the cells for 2 hours at 37C if added DNA plasmid have antibiotic resistance other than ampicillin.
- Prepare and Label two plastic plates with LB and appropriate antibiotic.
- Plate 300 ul of the culture onto first dish and spread.
- Add 900 ul of LB to 100 ul of the culture and plate 300 ul of it onto second dish and spread.
- Incubate the plates at 37C for 16~20 hours.
Mini-prep
We are using mini-prep kit of Nippon genetics: FastGene Plasmid mini kit. This kit contains these reagents and wares: mP1, mP2, mP3, mP4, mP5, mP6, column and collection tube.
- Centrifuge 1~5 ml of culture at over 10,000 rpm for 2 min.
- Remove the supernatant.
- Add 200ul of mP1 then voltexing.
- Add 200ul of mP2 and invert the tube then leave for 2 min at room temperature.
- Add 200ul of mP3 then invert the tube.
- Centrifuge at 1,3000 rpm for 8 min.
- Centrifuge 1,3000 rpm for 1 min to load supernatant from column to collection tube.
- Remove filtrate and add 400 ul of mP4 then centrifuge 13,000 rpm for 1 min.
- Remove filtrate and add 600 ul of mP5 then centrifuge 13,000 rpm for 1 min.
- Remove filtrate and centrifuge 13,000 rpm for 2 min.
- Set column into 1.5 ml centrifuge tube.
- Add 50 ul of mP6.
- Centrifuge at 13,000 rpm for 2 min.