Team:TzuChiU Formosa/Notebook

From 2010.igem.org

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[[Image:TzuChiU_Formosa_team.png|600px|Your team picture]]
+
==Protocol==
 +
===PCR (total volume: 50 μl )===
 +
:* 25 μl Green master mix (Promega)
 +
:* 2 μl each primer
 +
:* 2 μl template
 +
:* 19 μl ddH2O
 +
:* Cycle 95/2 min; 30x(95/1 min;56/1 min;72/2 min); 72/7 min
-
==Notebook==
+
===Gel Extraction- use Gel /PCR DNA Fragments Extraction Kit (Geneaid)===
 +
:* Loading the PCR products into agarose gel; run gel with 1X TAE buffer
 +
:* Excise the agarose gel slice containing relevant DNA fregments
 +
:* Transfer gel slice to a 1.5 ml microcentrifuge tube
 +
:* Add 500 μl DF buffer; incubate at 56 ℃ until gel slice has been completely dissolved
 +
:* Transfer 800 μl gel mixture to DF column
 +
:* Centrifuge 13000 rpm, 1 min
 +
:* Discard the flow-through and place the DF column back
 +
:* Add 400 μl W1 buffer; Centrifuge 13000 rpm, 1 min; Discard the flow-through
 +
:* Add 600 μl wash buffer; Centrifuge 13000 rpm, 1 min; Discard the flow-through
 +
:* Centrifuge 13000 rpm, 5 min; air- dry
 +
:* Add 50 μl ddH2O; Centrifuge 13000 rpm,1 min
 +
:* Storage at – 20℃
-
You should make use of the calendar feature on the wiki and start a lab notebook.  This may be looked at by the judges to see how your work progressed throughout the summer.  It is a very useful organizational tool as well.
+
===TA Cloning- use pGEM®-T Easy Vector (Promega)===
 +
:* 1μl pGEM®-T Easy Vector
 +
:* 5μl 2X rapid ligation buffer
 +
:* 1μl T4 DNA ligase
 +
:* 3μl insert
 +
:* 4 ℃, overnight
-
== '''Team 2: Poseidon - Save the World''' ==
+
===Ligation on pSB1C3- use Elite T4 DNA ligase (Geneaid)===
 +
:* 9 μl insert
 +
:* 3 μl vector
 +
:* 2 μl 10X ligation buffer A
 +
:* 2 μl 10X ligation buffer B
 +
:* 1 μl Elite T4 DNA ligase
 +
:* 3 μl ddH2O
 +
:* 16℃, overnight
-
Day 1: Sep 1 ,2010
+
===Transformation===
 +
:* Take out XL-10 Gold competent cell from -80 ℃ refrigerator; put on ice
 +
:* Add 2 μl GFP generator DNA solution into competent cell
 +
:* Place on ice, 30 min
 +
:* Heat shock, 42 ℃, 90 sec
 +
:* Chill on ice, 5 min
 +
:* Add 800 μl LB medium (without antibiotics)
 +
:* Incubate at 37 ℃, 1 hr
 +
:* For TA ligation product, smear 100 μl 50 mg /ml Ampicilin, 100 μl 0.1 M IPTG and 50 μl X-gal over LB agar plate.For pSB1C3 ligation product, smear 100μl 34 mg /ml Chlorophenicol over the plate.
 +
:* Bacteria solution centrifuge 3000 rpm, 5min
 +
:* Remove 700 μl supernatant
 +
:* Mix bacteria solution; Smear 100 μl over LB agar plate
 +
:* Incubate at 37 ℃, overnight
-
* Primer design:
+
===Plasmid Extraction===
 +
:* Pick single colonies; each colony incubate with 3 ml LB medium (add antibiotics); 37℃, overnight
 +
:* Centrifuge 7500 rpm, 1 min; remove supernatant
 +
:* Resuspend bacteria with 200 μl PD1 buffer; resuspend bacterial pellet
 +
:* Add 200 μl PD2 buffer; gently mix
 +
:* Add 300 μl PD3 buffer; gently mix
 +
:* Centrifuge 13000 rpm, 15 min
 +
:* Transfer supernatant to kit filter
 +
:* Add 400 μl W1 buffer; Centrifuge 13000 rpm, 1 min; Discard the flow-through
 +
:* Add 600 μl Wash buffer; Centrifuge 13000 rpm, 1 min; Discard the flow-through
 +
:* Centrifuge 13000 rpm, 5 min; air- dry
 +
:* Dissolve DNA with 50 μl ddH2O; Centrifuge 13000 rpm,1 min
 +
:* Storage at – 20℃
-
{|border = "0"
+
===Double Enzyme Digestion===
-
|-
+
:* 16 μl ddH2O
-
|rowspan="3"|
+
:* 0.5 μl Enzyme 1
-
 
+
:* 0.5 μl Enzyme 2
-
|
+
:* 3 μl NEB 10X buffer
-
<gallery>
+
:* 0.3 μl NEB 10X BSA
-
Image:TzuChiU_Formosa_Primer design.png|Primer design
+
:* 10 μl DNA
-
</gallery>
+
:* Incubate at 37 ℃, 3 hr
-
|}
+
-
 
+
-
* Note:
+
-
 
+
-
# For all of our construct assembly ,we designed XbaⅠrestriction site at 5’ end and SpeⅠat 3’ end.<br>
+
-
# We design the plus strain & minus strain of RBS (BBa_B0034), Biobrick cloning site prefix, and suffix, then we can anneal complementary single strain into double strain fragments. There is an additional adenine at 3' end of each single strain, it is designed for ligation on pGEM®-T Easy Vector (Promega).<br>
+
-
 
+
-
 
+
-
Day 2: Sep 2 ,2010
+
-
 
+
-
* Order primers
+
-
 
+
-
 
+
-
Day 3: Sep 3 ,2010
+
-
 
+
-
* Cyanobacteria ATCC 51142 has arrived!!
+
-
* Take out GFP generator (BBa_E0240) from biobrick kit plate 1(site:12M):
+
-
# dilute DNA with 10 μl ddH2O<br>
+
-
# pipette mix DNA<br>
+
-
# transfer DNA solution to new eppendorf<br>
+
-
# storage at -20℃<br>
+
-
* Transform GFP generator (BBa_E0240) into XL-10 Gold competent cell:
+
-
# Take out XL-10 Gold competent cell from -80℃ refrigerator; put on ice<br>
+
-
# Add 2 μl GFP generator DNA solution into competent cell<br>
+
-
# Place on ice, 30 min<br>
+
-
# Heat shock, 42℃, 90 sec<br>
+
-
# Chill on ice, 5 min<br>
+
-
# Add 800 μl LB medium (without antibiotics)<br>
+
-
# Incubate at 37℃, 1 hr<br>
+
-
# bacteria solution centrifuge 3000 rpm, 5 min<br>
+
-
# Remove 700 μl supernatant<br>
+
-
# Smear 100 μl Ampicilin (50 mg/ml) over LB agar plate<br>
+
-
# Mix bacteria solution; Smear 100 μl over LB agar plate<br>
+
-
# Incubate at 37℃, overnight<br>
+
-
 
+
-
 
+
-
Day 4: Sep 4 ,2010
+
-
 
+
-
* Mini-prep : GFP generator (BBa_E0240) transformants
+
-
# Pick 4 colonies from GFP generator transformants<br>
+
-
# Each colony incubate with 1.5 ml LB medium (with Ampicilin (50mg/l)); 37 overnight<br>
+
-
 
+
-
 
+
-
Day 5: Sep- 5 ,2010
+
-
 
+
-
* Plasmid extraction (Homemade) : GFP generator (BBa_E0240) transformants
+
-
# Centrifuge 3000 rpm, 10 min; remove supernatant<br>
+
-
# Resuspend bacteria with 100 μl Solution Ⅰ<br>
+
-
# Add 200 μl Solution Ⅱ; gently mix<br>
+
-
# Add 150 μl Solution Ⅲ; gently mix<br>
+
-
# Centrifuge 13000 rpm, 10 min<br>
+
-
# Transfer supernatant to new 1.5 ml microcentrifuge tube<br>
+
-
# Add 1 ml 100% EtOH<br>
+
-
# Centrifuge 13000 rpm, 15 min<br>
+
-
# Remove supernatant<br>
+
-
# Wash pellet 1 ml 75% EtOH; remove supernatant; air-dry DNA pellet<br>
+
-
# Dissolve DNA pellet with 50 μl ddH2O<br>
+
-
* GFP generator (BBa_E0240) enzyme digestion (check)
+
-
# 6 μl RNase<br>
+
-
# 0.5 μl EoRⅠ<br>
+
-
# 0.5 μl PstⅠ<br>
+
-
# 0.3 μl BSA<br>
+
-
# 10 μl DNA<br>
+
-
# Incubate at 37℃, 3 hr <br>
+
-
 
+
-
[[Image:2010.09.05.png|300px|2010.09.05]]
+
-
 
+
-
 
+
-
Day 6: Sep 6 ,2010
+
-
 
+
-
* Primers have arrived!!
+
-
# Dilute primers to 100μM/μl; Take out 10 μl, add 90 μl ddH2O (dilute to 10μM/μl)<br>
+
-
 
+
-
 
+
-
Day 7: Sep 7 ,2010
+
-
 
+
-
* PCR (total volume: 25 μl ): alkB1, alkG, alkH, alkJ and GFP generator (BBa_E0240) genes
+
-
# 12.5 μl Green master mix (Promega)<br>
+
-
# 1 μl each primer<br>
+
-
# 1 μl template<br>
+
-
# 9.5 μl ddH2O<br>
+
-
# cycle 95/2 min; 30x(95/1 min; 56/2 min; 72/1.5 min); 72/7 min<br>
+
-
 
+
-
[[Image:2010.09.07.png|300px|2010.09.07]]
+
-
 
+
-
* Gel extraction: alkB1, alkG, alkH, alkJ and GFP generator (BBa_E0240) genes
+
-
- use Gel/PCR DNA Fragments Extraction Kit (Geneaid)<br>
+
-
# Loading the PCR products into agarose gel; run gel with 1X TAE buffer<br>
+
-
# Excise the agarose gel slice containing relevant DNA fragments <br>
+
-
# Transfer gel slice to a 1.5 ml microcentrifuge tube<br>
+
-
# Add 500 μl DF buffer; incubate at 56℃ until gel slice has been completely dissolved<br>
+
-
# Transfer 800 μl gel mixture to DF column<br>
+
-
# Centrifuge 13000 rpm, 1 min<br>
+
-
# Discard the flow-through and place the DF column back<br>
+
-
# Add 400 μl W1 buffer; Centrifuge 13000 rpm, 1 min; Discard the flow-through<br>
+
-
# Add 600 μl wash buffer; Centrifuge 13000 rpm, 1 min; Discard the flow-through<br>
+
-
# Centrifuge 13000 rpm, 5 min; air-dry<br>
+
-
# Add 50 μl ddH2O; Centrifuge 13000 rpm,1 min<br>
+
-
# Storage at –20℃<br>
+
-
* TA ligation: alkB1, alkG, alkH, alkJ and GFP generator (BBa_E0240) genes
+
-
- use pGEM®-T Easy Vector (Promega)<br>
+
-
# 1μl pGEM®-T Easy Vector<br>
+
-
# 5μl 2X rapid ligation buffer<br>
+
-
# 1μl T4 DNA ligase<br>
+
-
# 3μl insert<br>
+
-
# 4℃, overnight<br>
+
-
 
+
-
 
+
-
Day 8: Sep 8 ,2010
+
-
 
+
-
* Transformation
+
-
- transform alkB1, alkG, alkH, alkJ and GFP generator (BBa_E0240) TA ligation products into XL-10 Gold competent cell<br>
+
-
 
+
-
 
+
-
Day 9: Sep 9 ,2010
+
-
 
+
-
* Annealing
+
-
- anneal complementary single strain of RBS (BBa_B0034), Biobrick cloning site prefix (BBa_G00000) and Biobrick cloning site suffix (BBa_G00001) into double strain fragments<br>
+
-
# dilute oligo primers to 10 μM/μl<br>
+
-
# add 12.5 μl plus strain<br>
+
-
# add 12.5 μl minus strain<br>
+
-
# Heat to 95℃; cool down to room temperature slowly <br>
+
-
* TA ligation
+
-
- Ligate RBS (BBa_B0034) , Biobrick cloning site prefix (BBa_G00000) and Biobrick cloning site suffix (BBa_G00001) on pGEM®-T Easy Vector (Promega)<br>
+
-
# 1μl pGEM®-T Easy Vector<br>
+
-
# 5μl 2X rapid ligation buffer<br>
+
-
# 1μl T4 DNA ligase<br>
+
-
# 3μl insert<br>
+
-
# 4℃, overnight<br>
+
-
* Mini-prep
+
-
- Pick alkB1, alkG, alkH, alkJ and GFP generator (BBa_E0240) transformants (ligate on pGEM-T easy vector)<br>
+
-
# Pick 4 colonies from alkB1, alkG, alkH, alkJ and GFP generator transformants<br>
+
-
# Each colony incubate with 1.5 ml LB medium (with Ampicilin (50mg/l)); 37℃, overnight<br>
+
-
 
+
-
 
+
-
Day10: Sep 10 ,2010
+
-
 
+
-
* Plasmid extraction (Homemade) : alkB1, alkG, alkH, alkJ and GFP generator (BBa_E0240) transformants (ligate on pGEM-T easy vector)
+
-
* Enzyme digestion (check)
+
-
# 6 μl RNase <br>
+
-
# 3 μl NEB buffer 4<br>
+
-
# 0.5 μl XbaⅠ<br>
+
-
# 0.5 μl SpeⅠ<br>
+
-
# 10 μl plasmid DNA<br>
+
-
# Incubate at 37℃, 3 hr<br>
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
* Transformation
+
-
- transform RBS (BBa_B0034), Biobrick cloning site prefix (BBa_G00000) and Biobrick cloning site suffix (BBa_G00001) into XL-10 Gold competent cell<br>
+
-
 
+
-
 
+
-
Day11: Sep 11 ,2010
+
-
 
+
-
* Mini-prep
+
-
- Pick RBS (BBa_B0034), Biobrick cloning site prefix (BBa_G00000) and Biobrick cloning site suffix (BBa_G00001) transformants (ligate on pGEM-T easy vector)<br>
+
-
# Pick 4 colonies from RBS , Biobrick cloning site prefix and Biobrick cloning site suffix  transformants<br>
+
-
# Each colony incubate with 1.5 ml LB medium (with Ampicilin (50mg/l)); 37℃, overnight<br>
+
-
- alkB1, alkG, alkH, alkJ and GFP generator (BBa_E0240) (have been checked in correct size and ligate on pGEM-T easy vector) mini-prep for plasmid extraction (use plasmid extraction Kit)<br>
+
-
# 5 μl bacterial stock<br>
+
-
# 10 ml LB (with Ampicilin (50mg/l))<br>
+
-
# Incubate at 37℃, overnight<br>
+

Latest revision as of 07:11, 4 December 2010

Contents

Protocol

PCR (total volume: 50 μl )

  • 25 μl Green master mix (Promega)
  • 2 μl each primer
  • 2 μl template
  • 19 μl ddH2O
  • Cycle 95/2 min; 30x(95/1 min;56/1 min;72/2 min); 72/7 min

Gel Extraction- use Gel /PCR DNA Fragments Extraction Kit (Geneaid)

  • Loading the PCR products into agarose gel; run gel with 1X TAE buffer
  • Excise the agarose gel slice containing relevant DNA fregments
  • Transfer gel slice to a 1.5 ml microcentrifuge tube
  • Add 500 μl DF buffer; incubate at 56 ℃ until gel slice has been completely dissolved
  • Transfer 800 μl gel mixture to DF column
  • Centrifuge 13000 rpm, 1 min
  • Discard the flow-through and place the DF column back
  • Add 400 μl W1 buffer; Centrifuge 13000 rpm, 1 min; Discard the flow-through
  • Add 600 μl wash buffer; Centrifuge 13000 rpm, 1 min; Discard the flow-through
  • Centrifuge 13000 rpm, 5 min; air- dry
  • Add 50 μl ddH2O; Centrifuge 13000 rpm,1 min
  • Storage at – 20℃

TA Cloning- use pGEM®-T Easy Vector (Promega)

  • 1μl pGEM®-T Easy Vector
  • 5μl 2X rapid ligation buffer
  • 1μl T4 DNA ligase
  • 3μl insert
  • 4 ℃, overnight

Ligation on pSB1C3- use Elite T4 DNA ligase (Geneaid)

  • 9 μl insert
  • 3 μl vector
  • 2 μl 10X ligation buffer A
  • 2 μl 10X ligation buffer B
  • 1 μl Elite T4 DNA ligase
  • 3 μl ddH2O
  • 16℃, overnight

Transformation

  • Take out XL-10 Gold competent cell from -80 ℃ refrigerator; put on ice
  • Add 2 μl GFP generator DNA solution into competent cell
  • Place on ice, 30 min
  • Heat shock, 42 ℃, 90 sec
  • Chill on ice, 5 min
  • Add 800 μl LB medium (without antibiotics)
  • Incubate at 37 ℃, 1 hr
  • For TA ligation product, smear 100 μl 50 mg /ml Ampicilin, 100 μl 0.1 M IPTG and 50 μl X-gal over LB agar plate.For pSB1C3 ligation product, smear 100μl 34 mg /ml Chlorophenicol over the plate.
  • Bacteria solution centrifuge 3000 rpm, 5min
  • Remove 700 μl supernatant
  • Mix bacteria solution; Smear 100 μl over LB agar plate
  • Incubate at 37 ℃, overnight

Plasmid Extraction

  • Pick single colonies; each colony incubate with 3 ml LB medium (add antibiotics); 37℃, overnight
  • Centrifuge 7500 rpm, 1 min; remove supernatant
  • Resuspend bacteria with 200 μl PD1 buffer; resuspend bacterial pellet
  • Add 200 μl PD2 buffer; gently mix
  • Add 300 μl PD3 buffer; gently mix
  • Centrifuge 13000 rpm, 15 min
  • Transfer supernatant to kit filter
  • Add 400 μl W1 buffer; Centrifuge 13000 rpm, 1 min; Discard the flow-through
  • Add 600 μl Wash buffer; Centrifuge 13000 rpm, 1 min; Discard the flow-through
  • Centrifuge 13000 rpm, 5 min; air- dry
  • Dissolve DNA with 50 μl ddH2O; Centrifuge 13000 rpm,1 min
  • Storage at – 20℃

Double Enzyme Digestion

  • 16 μl ddH2O
  • 0.5 μl Enzyme 1
  • 0.5 μl Enzyme 2
  • 3 μl NEB 10X buffer
  • 0.3 μl NEB 10X BSA
  • 10 μl DNA
  • Incubate at 37 ℃, 3 hr
Retrieved from "http://2010.igem.org/Team:TzuChiU_Formosa/Notebook"