Team:TzuChiU Formosa/Protocol

From 2009.igem.org

(Difference between revisions)

Revision as of 13:25, 20 October 2009

Protocol

Tansformation Protocol

Take the cp919 competent cell in an eppendorf tube from -80℃ freezer put in ice.
Add 4ul plasmid to competent cell and place in ice for 30 minutes.
Put the transformed cells into 42℃ water bath for 90 seconds.
Then place the cells in ice for 2 minutes.
Add 1ml LB to the cells and mixed.
Put the eppendorf tube in 37℃ water bath and incubate for an hour.
Spin down at 7000 rpm for 5 minutes and remove most of the supernatant.
While the cells are incubated at 37℃ water bath spread 100ul Ampicilin(50mg/ml) on the plate. When the plate is dried, spread bacteria on the plate.
Incubate at 37℃ incubater for 16~18 hours.

Competent Cell (CP919-Cph8)

Day1. Streak out the E.coli strain on an LB plate (with kanamycin)and incubate at 37℃ overnight (16-20 hours).
Day2. Select a single colony and inoculate 10 ml sterile LB and grow overnight (16-20 hours) in a 37℃ shaker incubator.
Day3. Add 2ml overnight culture to 250ml flask containing 100 ml LB.
Grow the cultures to OD600 = 0.2~0.4 (incubate with shaking for 75~90 min.)
Spin down the bacteria at 4℃,3000 rpm for 10 min.
Discard the supernatant and mix the cell pellet with 10ml FSB.
Keep the cells on ice for 3~4 hours.
Spin down, at 4℃,3000 rpm for 10 min.
Discard the supernatant and mix the cell pellet with 5ml FSB.
Pipet 200μl of the cell suspension into sterile 1.5ml eppendorf tubes. Freeze these tubes in liquid nitrogen, then transfer them to -80℃ freezer.

Real Time PCR

1. Take ice for preparing.

2. dilution of concentration：

2.1

template DNA（Omp R）：500 times diluted

499μl ddH2O ＋ 1μl omp R＝500μl

2.2

primer forward ＆ primer reverse（p88）：10 times diluted

10μl p88 primer forward＋ 90μl ddH2O

3. Materials：

 Template DNA（10ng/μl）		5
 10× PCR buffer			2
 10× dNTP（2mM）			2
 forward primer（10μM）		0.5
 reverse primer（10μM）		0.5
 Pfu DNA polymerase（2Kb）	0.1
 PCR water			9.9
 _______________________________________
 Total				20 μl

4. Step：

Meacure template DNA 2μl Measure 10× PCR buffer 2μl Measure 10× dNTP 2μl Measure forward primer 0.5μl Measure reverse primer 0.5μl Measure Pfu DNA polymerase 0.1μl Follow this step ，puttng the reagent in the eppendorf.

5. Use the PCR to amplify our product：PCR program

5.1

94℃ 30 seconds

60℃ 30seconds

72℃ 2 minutes

Cycle 9 times

5.2

94℃ 30 seconds

55℃ 30seconds

72℃ 2 minutes

Cycle 34 times

72℃ 10 minutes

6. The PCR production separate by 1％ agar.

7. After separate using the EtBr to dye agar and exposing by UV.

T-A cloning protocol

Take an eppendorf,and add these one by one,after mix well,overnight 4℃.

 2x ligase buffer	7.5μl
 Insert(Aeq.-GFP)	5.5μl
 Vector(pGEM-T-easy)	1μl
 T4 DNA exp 3/12	1μl
 _________________________________
 total			15μl

Cloning PCR

Add 10μl cell, centrifuge 14000 rpm, 10 min
Discard the supernatant
Add 500μl ddH20, Votex
Boiling 20min, 100℃
Add 5μl DNA, 10x dNTP 2μl, 10x Buffer 2μl, forward primer（10μM）0.5μl reverse primer（10μM）0.5μl, Taq DNA polymerase 0.1μl, ddH20
Use the PCR to amplify our product：PCR program

 95℃ 4 min 
 94℃ 30seconds 
 55℃ 40seconds 
 72℃ 2 min
 Cycle 34 times
 25℃ 2 min

　7. The PCR production separate by 1％ agar.

　8. After separate using the EtBr to dye agar and exposing by UV.

@@ Line 17: / Line 17: @@
 ====Tansformation Protocol ====
-#Take the cp919 competent cell eppendorf from -80℃ freezer.
+#Take the cp919 competent cell in an eppendorf tube from -80℃ freezer put in ice.
-#Add 4ul plasmid to competent cell and place at ice for 30 minutes.
+#Add 4ul plasmid to competent cell and place in ice for 30 minutes.
-#Put the eppendorf into 42℃ water bath for 90 seconds.
+#Put the transformed cells into 42℃ water bath for 90 seconds.
-#Place the eppendorf at ice for 2 minutes.
+#Then place the cells in ice for 2 minutes.
-#Add 1ml LBM to the eppendorf and well mixed.
+#Add 1ml LB to the cells and mixed.
-#Put the eppendorf into 37℃ water bath incubate for an hour.
+#Put the eppendorf tube in 37℃ water bath and incubate for an hour.
-#Centrifuged at 7000 r.p.m for 5 minutes and remove some supernatant liquid.
+#Spin down at 7000 rpm for 5 minutes and remove most of the supernatant.
-#Spread antibiotic which we need on plate. When the plate dry, spread appropriate the culture liquid on plate.
+#While the cells are incubated at 37℃ water bath spread 100ul Ampicilin(50mg/ml) on the plate. When the plate is dried, spread bacteria on the plate.
-#Incubate into 37℃ water bath for 16~18 hours.
+#Incubate at 37℃ incubater for 16~18 hours.
 ====Competent Cell (CP919-Cph8)====
-#Day1. Streak out the E.coli strain on an LBM plate (with kanamycin antibiotic) to isolate colonies and incubate at 37℃ overnight (16-20 hours).
+#Day1. Streak out the E.coli strain on an LB plate (with kanamycin)and incubate at 37℃ overnight (16-20 hours).
-#Day2. Select a single colony and inoculate 10 ml sterile LBM Grow overnight (16-20 hours) in a 37℃ shaker incubator.
+#Day2. Select a single colony and inoculate 10 ml sterile LB and grow overnight (16-20 hours) in a 37℃ shaker incubator.
-#Day3. Add 2ml overnight culture to 250ml flask which with 100 ml sterile LBM.
+#Day3. Add 2ml overnight culture to 250ml flask containing 100 ml LB.
-#Grow the cultures to OD600 = 0.2~0.4 (incubate about 75~90 min.).
+#Grow the cultures to OD600 = 0.2~0.4 (incubate with shaking for 75~90 min.)
-#Centrifuge, 4℃,3000 r.p.m,10 min.
+#Spin down the bacteria at 4℃,3000 rpm for 10 min.
 #Discard the supernatant and mix the cell pellet with 10ml FSB.
 #Keep the cells on ice for 3~4 hours.
-#Centrifuge, 4℃,3000 r.p.m,10 min.
+#Spin down, at 4℃,3000 rpm for 10 min.
 #Discard the supernatant and mix the cell pellet with 5ml FSB.
-#Pipet 200μl of the cell suspension into sterile 1.5ml eppendorf. Freeze these tubes on N2 (aq.) and then transfer them to -80℃ freezer.
+#Pipet 200μl of the cell suspension into sterile 1.5ml eppendorf tubes. Freeze these tubes in liquid nitrogen, then transfer them to -80℃ freezer.