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
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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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{|
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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 &ge; 63)
2STEP Cycle (Tm value &ge; 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 &le; 63)
3STEP Cycle (Tm value &le; 63)
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8~9 x 15~30
8~9 x 15~30
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Revision as of 18:30, 26 September 2012

Contents

General Protocols

Transformation

  1. Add 1~2 ul of DNA to 50 ul of thawed competent cells on ice.
  2. Incubate on ice for 30 min.
  3. Add 600 ul of LB.
  4. Incubate the cells for 2 hours at 37C if added DNA plasmid have antibiotic resistance other than ampicillin.
  5. Prepare and Label two plastic plates with LB and appropriate antibiotic.
  6. Plate 300 ul of the culture onto first dish and spread.
  7. Add 900 ul of LB to 100 ul of the culture and plate 300 ul of it onto second dish and spread.
  8. 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.

  1. Centrifuge 1~5 ml of culture at over 10,000 rpm for 2 min.
  2. Remove the supernatant.
  3. Add 200 ul of mP1 then voltexing.
  4. Add 200 ul of mP2 and invert the tube then leave for 2 min at room temperature.
  5. Add 200 ul of mP3 then invert the tube.
  6. Centrifuge at 1,3000 rpm for 8 min.
  7. Centrifuge 1,3000 rpm for 1 min to load supernatant from column to collection tube.
  8. Remove filtrate and add 400 ul of mP4 then centrifuge 13,000 rpm for 1 min.
  9. Remove filtrate and add 600 ul of mP5 then centrifuge 13,000 rpm for 1 min.
  10. Remove filtrate and centrifuge 13,000 rpm for 2 min.
  11. Set column into 1.5 ml centrifuge tube.
  12. Add 50 ul of mP6.
  13. Centrifuge at 13,000 rpm for 2 min.

Ethanol precipitation

  1. Add 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
  2. Centrifuge in 15000 rpm for 10~15 min at 4C.
  3. Remove supernatant and added 220 ul of 70% ethanol.
  4. Centrifuge in 15000 rpm for 5~15 min at 4C.
  5. Remove supernatant and air drying in room temperature
  6. 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
  1. Put gel into gel box.
  2. Add 1/2 TBE buffer
  3. Add 5ul of EtBr.
  4. Pre-migration for 30 min.
  5. Add appropriate DNA solution and ladder.
  6. 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.

  1. Add 500 ul of GP1 to ~300mg of migrated gel and vortexing.
  2. Incubate the mixture at 55C for 10~15 min and invert.
  3. Load the sample onto the column at 13,000 rpm for 1 min.
  4. Remove filtrate and Add 600 ul of GP2 and centrifuge at 13,000 rpm for 1 min.
  5. Remove filtrate and Add 600 ul of GP2 and centrifuge at 13,000 rpm for 1 min again.
  6. Remove filtrate and Centrifuge at 1,3000 rpm for 2 min.
  7. Set column into 1.5 ml centrifuge tube.
  8. Add 50 ul of GP6.
  9. 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


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