Team:HokkaidoU Japan/Notebook/overall protocols
From 2012.igem.org
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==General Protocols== | ==General Protocols== | ||
===Transformation=== | ===Transformation=== | ||
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#Incubate the plates at 37C for 16~20 hours. | #Incubate the plates at 37C for 16~20 hours. | ||
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===Mini-prep=== | ===Mini-prep=== | ||
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#Centrifuge at 13,000 rpm for 2 min. | #Centrifuge at 13,000 rpm for 2 min. | ||
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===Ethanol precipitation=== | ===Ethanol precipitation=== | ||
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#add 10 ul of DW. | #add 10 ul of DW. | ||
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===Ligation=== | ===Ligation=== | ||
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|10 ul | |10 ul | ||
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Ligation reaction time was in detail below. | Ligation reaction time was in detail below. | ||
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===Digestion=== | ===Digestion=== | ||
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===Electrophoresis=== | ===Electrophoresis=== | ||
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Preparing following 1/2 TBE buffer and 1~2% agarose gel. | Preparing following 1/2 TBE buffer and 1~2% agarose gel. | ||
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|1/2 TBE Buffer composition | |1/2 TBE Buffer composition | ||
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|200 ml | |200 ml | ||
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#Put gel into gel box. | #Put gel into gel box. | ||
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#Add appropriate DNA solution and ladder. | #Add appropriate DNA solution and ladder. | ||
#Run the gel. | #Run the gel. | ||
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Note:Almost all of electrophoresis images were inverted to observe the results more clearer. | Note:Almost all of electrophoresis images were inverted to observe the results more clearer. | ||
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===Gel extraction=== | ===Gel extraction=== | ||
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#Centrifuge at 13,000 rpm for 2 min. | #Centrifuge at 13,000 rpm for 2 min. | ||
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===PCR=== | ===PCR=== | ||
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PCR is high-speed amplification method of DNA. We use KOD-Plus-Neo (TOYOBO) as polymerase. | PCR is high-speed amplification method of DNA. We use KOD-Plus-Neo (TOYOBO) as polymerase. | ||
Mixed PCR solutions and run the PCR machine in a program which is detailed below. | Mixed PCR solutions and run the PCR machine in a program which is detailed below. | ||
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2STEP Cycle (Tm value ≥ 63) | 2STEP Cycle (Tm value ≥ 63) | ||
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Cycle:2~4 x 25~45 | Cycle:2~4 x 25~45 | ||
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3STEP Cycle (Tm value ≤ 63) | 3STEP Cycle (Tm value ≤ 63) | ||
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8~9 x 15~30 | 8~9 x 15~30 | ||
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{{Team:HokkaidoU_Japan/footer}} | {{Team:HokkaidoU_Japan/footer}} |
Revision as of 18:30, 26 September 2012
Contents |
General Protocols
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 use 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 200 ul of mP1 then voltexing.
- Add 200 ul of mP2 and invert the tube then leave for 2 min at room temperature.
- Add 200 ul 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.
Ethanol precipitation
- Add 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
- Centrifuge in 15000 rpm for 10~15 min at 4C.
- Remove supernatant and added 220 ul of 70% ethanol.
- Centrifuge in 15000 rpm for 5~15 min at 4C.
- Remove supernatant and air drying in room temperature
- add 10 ul of DW.
Ligation
Mix the following reagents in 0.2 ml PCR tube. We use Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer.
Vector DNA | 1 ul |
Insert DNA | 2 ul |
DW | 2 ul |
Ligation Mighty Mix | 5 ul |
Total | 10 ul |
Ligation reaction time was in detail below.
Degree | Minute |
16 | 30 |
65 | 10 |
4 | Hold |
Digestion
Mix the following reagents in 0.2 ml PCR tube. Total volume is over 10ul.
DNA solution |
appropriate Restriction enzyme |
appropriate buffer |
DW |
Run a following program in PCR machine.
Degree | Minute |
37 | 59 |
37 | 59 |
37 | 2 |
65(inactivation) | 15 |
4 | Hold |
Electrophoresis
Preparing following 1/2 TBE buffer and 1~2% agarose gel.
1/2 TBE Buffer composition | |
Tris amino methane | 108 g |
Boric acid | 55 g |
0.5 M EDTA(pH8.0) | 40 ml |
Total | 20 L |
Agarose gel composition | |
Agarose | 1~2 g |
1/2 TBE buffer | 200 ml |
- Put gel into gel box.
- Add 1/2 TBE buffer
- Add 5ul of EtBr.
- Pre-migration for 30 min.
- Add appropriate DNA solution and ladder.
- Run the gel.
Note:Almost all of electrophoresis images were inverted to observe the results more clearer.
Gel extraction
We use Gel extraction kit of Nippon genetics: FastGene Gel/PCR Extraction kit. This kit contains these reagents and wares: GP1, GP2, GP3, column and collection tube.
- Add 500 ul of GP1 to ~300mg of migrated gel and vortexing.
- Incubate the mixture at 55C for 10~15 min and invert.
- Load the sample onto the column at 13,000 rpm for 1 min.
- Remove filtrate and Add 600 ul of GP2 and centrifuge at 13,000 rpm for 1 min.
- Remove filtrate and Add 600 ul of GP2 and centrifuge at 13,000 rpm for 1 min again.
- Remove filtrate and Centrifuge at 1,3000 rpm for 2 min.
- Set column into 1.5 ml centrifuge tube.
- Add 50 ul of GP6.
- Centrifuge at 13,000 rpm for 2 min.
PCR
PCR is high-speed amplification method of DNA. We use KOD-Plus-Neo (TOYOBO) as polymerase. Mixed PCR solutions and run the PCR machine in a program which is detailed below.
Solution | Volume(ul) |
DNA | 1 |
Forward primer | 1 |
Reverse primer | 1 |
MgSO4 | 3 |
dNTP | 5 |
10x KOD-Plus-Neo Buffer | 5 |
KOD-Plus-Neo | 1 |
DW | 33 |
Total | 50 |
2STEP Cycle (Tm value ≥ 63)
Number | Degree | Second |
1 | 94 | 120 |
2 | 98 | 10 |
3 | 68 | 30/kb |
4 | 68 | 30/kbp |
5 | 4 | HOLD |
Cycle:2~4 x 25~45
3STEP Cycle (Tm value ≤ 63)
Number | Degree | Second |
1 | 94 | 120 |
2 | 98 | 10 |
3 | Tm value of primer | 30 |
4 | 68 | 30/kbp |
5 | 4 | HOLD |
Cycle:2~4 x 25~45
Step-down Cycle (If there were so many extra-band and smear)
Number | Degree | Second |
1 | 94 | 120 |
2 | 98 | 10 |
3 | 74 | 30/kb |
4 | 98 | 10 |
5 | 72 | 30/kb |
6 | 98 | 10 |
7 | 70 | 30/kb |
8 | 98 | 10 |
9 | 68 | 30/kb |
10 | 68 | 420 |
11 | 4 | HOLD |
Cycle:2~3 x 5; 4~5 x 5; 6~7 x 5; 8~9 x 15~30