Team:TMU-Tokyo/Notebook/Experiment/1st week (8.13 - 8.19)
From 2012.igem.org
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+ | <b>■ 14th Aug</b><br /> | ||
+ | (Construction of Device3) | ||
+ | <b>・PCR of vector (BBa_K299009) and insert (fdh4AB)</b> | ||
+ | ・Preparation of 1.5 % Agarose Gel Electrophoresis<Br> | ||
+ | →Failure: because of Preparation of Gel protocol miss<Br> | ||
+ | <Br> | ||
+ | ・Preparation of 1.5 % Agarose Gel Electrophoresis (again)<Br> | ||
+ | →Failure: because of Preparation of Gel protocol miss<Br> | ||
+ | <Br> | ||
+ | ・Preparation of 1.5 % Agarose Gel<Br> | ||
+ | →Failure: because of PCR protocol miss<Br> | ||
+ | <Br> | ||
+ | <b>・PCR of vector (BBa_K299009) and insert (fdh4AB)</b> | ||
+ | ・Preparation of 0.8 % Agarose Gel | ||
+ | ・Correction of the PCR protocol and Preparation of Gel protocol<Br> | ||
+ | <Br> | ||
+ | <Br> | ||
<b>■15th Aug</b><br /> | <b>■15th Aug</b><br /> | ||
+ | <Br> | ||
+ | (Construction of Device3)<Br> | ||
+ | ・Electrophoresis of previous sample<Br> | ||
+ | →Failure: because of PCR protocol miss<Br> | ||
+ | <b>・PCR of vector (BBa_K299009) and insert (fdh4AB)</b><Br> | ||
+ | ・Preparation of 0.8 % Agarose Gel Electrophoresis<Br> | ||
+ | →Failure: because of smear<Br> | ||
+ | <b>・PCR of vector (BBa_K299009)</b><Br> | ||
+ | <Br> | ||
+ | |||
+ | |||
+ | |||
+ | |||
<b>・Digestion</b><br /> | <b>・Digestion</b><br /> | ||
- | 1. Mixed following<br /> | + | 1. Mixed following: total 20µL<br /> |
- | DW 15µL / DNA solution 2µL / 10x L buffer 2µL / SacI 1µL;<Br | + | (DW 15µL / DNA solution 2µL / 10x L buffer 2µL / SacI 1µL;)<Br> |
- | + | ||
2. Incubated at 37 °C for 1 hour.<br /> | 2. Incubated at 37 °C for 1 hour.<br /> | ||
3. Heat inactivated at 65 °C for 10 minutes.<br /> | 3. Heat inactivated at 65 °C for 10 minutes.<br /> | ||
- | 4. Mixed<br /> | + | 4. Mixed: Total 50µL<br /> |
- | DW 24µL / Reaction solution 20µL / 10x K buffer 5µL / BamHI 1µL;<Br | + | (DW 24µL / Reaction solution 20µL / 10x K buffer 5µL / BamHI 1µL;)<Br> |
- | + | ||
5. Incubated at 37 °C for 1 hour 40 minutes.<br /> | 5. Incubated at 37 °C for 1 hour 40 minutes.<br /> | ||
<br /> | <br /> | ||
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<br /> | <br /> | ||
<b>・Gel extraction</b><br /> | <b>・Gel extraction</b><br /> | ||
- | Excised the DNA fragment from an agarose gel. For each 100 mg of agarose gel added | + | Excised the DNA fragment from an agarose gel. For each 100 mg of agarose gel added 200µL Buffer NT.<br /> |
Incubated sample for 5 – 10 minutes at 50 C. Vortexed the sample briefly every 2 – 3 minuets until the gel slice was completely dissolved.<br /> | Incubated sample for 5 – 10 minutes at 50 C. Vortexed the sample briefly every 2 – 3 minuets until the gel slice was completely dissolved.<br /> | ||
- | Placed a NucleoSpin Extract II Columu into a Collection Tube and loaded the sample. Centrifuged for 30 seconds at 11, | + | Placed a NucleoSpin Extract II Columu into a Collection Tube and loaded the sample. Centrifuged for 30 seconds at 11,000x g. Discarded flow-through and placed the column backed into the collection tube.<br /> |
- | Added | + | Added 700µL Buffer NT3 to the NucleoSpin Extact II Column. Centrifuged for 30 s at 11,000x g. Discarded flow-through and placed the column backed into the collection tube.<br /> |
- | Centrifuged for 1 | + | Centrifuged for 1 min at 11,000x g to remove Buffer NT3 completely.<br /> |
- | Placed the NucleoSpin Extract II Column into a new 1.5 ml microcetrifuge tube. Added | + | Placed the NucleoSpin Extract II Column into a new 1.5 ml microcetrifuge tube. Added 30µL Buffer NE and incubated at room temperature for 1 min. Centrifuged for 1 min at 11,000x g.<br /> |
<br /> | <br /> | ||
<b>・Densitometry</b><br /> | <b>・Densitometry</b><br /> | ||
Diluted DNA samples 50 times with a solvent.<br /> | Diluted DNA samples 50 times with a solvent.<br /> | ||
Turned on the machine; GeneQuant 100.<br /> | Turned on the machine; GeneQuant 100.<br /> | ||
- | Poured the solvent | + | Poured the solvent 100µL into a cuvette and adjusted 0.<br /> |
Threw the solvent, poured the DNA sample.<br /> | Threw the solvent, poured the DNA sample.<br /> | ||
Measured the concentration. <br /> | Measured the concentration. <br /> | ||
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We could not obtain the target fragments of fdh4AB.<br /> | We could not obtain the target fragments of fdh4AB.<br /> | ||
<br /> | <br /> | ||
+ | <Br> | ||
<b>■16th Aug</b><br /> | <b>■16th Aug</b><br /> | ||
- | + | <Br> | |
+ | (Construction of Device3)<Br> | ||
+ | ・Preparation of 0.8 % Agarose Gel Electrophoresis<Br> | ||
+ | →Success: We observed appropriate bands!<Br> | ||
+ | ・PCR of insert (fdh4AB) | ||
+ | ・Check the fdh4AB primer’s sequence | ||
+ | <Br> | ||
+ | <Br> | ||
+ | <Br> | ||
+ | |||
+ | <b>・Refine of PCR products</b>: fdh4AB and Backbone plasmid<br /> | ||
Mixed 1 volume of sample with 2 volumes of Buffer NT.<br /> | Mixed 1 volume of sample with 2 volumes of Buffer NT.<br /> | ||
- | Placed a NucleoSpin Extract II Column into a Collection Tube and loaded the sample. Centrifuged for 30 | + | Placed a NucleoSpin Extract II Column into a Collection Tube and loaded the sample. Centrifuged for 30 s at 11,000x g. Discarded flow-through and placed the column back into the collection tube.<br /> |
- | Added | + | Added 700µL Buffer NT3 to the NucleoSpin Extract II Column. Centrifuged for 30 s at 11,000x g. Discarded flow-though and placed the column back into the collection tube.<br /> |
- | Centrifuged for 1 minute at 11, | + | Centrifuged for 1 minute at 11,000x g to remove Buffer NT3 completely.<br /> |
- | Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added | + | Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added 30µL Buffer NE and incubated at room temperature for 1 min. Centrifuged for 1 minute at 11,000x g.<br /> |
<br /> | <br /> | ||
<b>・Densitometry</b><br /> | <b>・Densitometry</b><br /> | ||
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Backbone plasmid No. 01 was 110 ng/ μl<br /> | Backbone plasmid No. 01 was 110 ng/ μl<br /> | ||
No. 02 was 60 ng/ μl<br /> | No. 02 was 60 ng/ μl<br /> | ||
+ | <Br> | ||
<b>・Digestion</b><br /> | <b>・Digestion</b><br /> | ||
- | Mixed following<br /> | + | 1. Mixed following<br /> |
- | Fdh4AB | + | Fdh4AB: total 25µL (milliQ 6.5µL / DNA solution 15µL / 10x L buffer 2.5µL / SacI 1µL)<br /> |
- | milliQ | + | Backbone plasmid: total 25µL (milliQ 1.5µL / DNA solution 20µL / 10x L buffer 2.5µL / SacI 1µL)<br /> |
- | DNA solution | + | |
- | + | ||
- | SacI | + | |
- | + | ||
- | <br /> | + | |
- | Backbone plasmid | + | |
- | milliQ | + | |
- | DNA solution | + | |
- | + | ||
- | SacI | + | |
- | + | ||
- | <br /> | + | |
2. Incubated for 1 hour at 37 °C.<br /> | 2. Incubated for 1 hour at 37 °C.<br /> | ||
3. Heat inactivated at 65 °C for 10 minutes.<br /> | 3. Heat inactivated at 65 °C for 10 minutes.<br /> | ||
- | 4. Mixed following | + | 4. Mixed following; total 50µL (milliQ 19µL / Reaction solution 25 µL / 10x K buffer 5µL / BamHI 1µL)<br /> |
- | milliQ | + | |
- | Reaction solution 25 | + | |
- | + | ||
- | BamHI | + | |
- | + | ||
- | <br /> | + | |
5. Incubated at 37 °C for 1 hour.<br /> | 5. Incubated at 37 °C for 1 hour.<br /> | ||
<br /> | <br /> | ||
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<Br> | <Br> | ||
<b>■17th Aug</b><br /> | <b>■17th Aug</b><br /> | ||
+ | <Br> | ||
+ | (Construction of Device3)<Br> | ||
+ | ・Preparation of 0.8 % Agarose Gel | ||
+ | ・Electrophoresis | ||
+ | →Failure: because of different bands | ||
+ | ・<b>PCR of insert (fdh4AB)</b> | ||
+ | ・Preparation of 0.8 % Agarose Ge<Br> | ||
+ | <Br> | ||
+ | |||
+ | |||
<b>Electrophoresis of PCR products</b>: before digestion of fdh4AB, after PCR products clean-up of fdh4AB, before PCR products clean-up of fdh4AB and diluted 100 times of PCR products of fdh4AB.<br /> | <b>Electrophoresis of PCR products</b>: before digestion of fdh4AB, after PCR products clean-up of fdh4AB, before PCR products clean-up of fdh4AB and diluted 100 times of PCR products of fdh4AB.<br /> | ||
- | |||
Put an agalose gel into the tank, and poured TBE buffer.<br /> | Put an agalose gel into the tank, and poured TBE buffer.<br /> | ||
Mixed DNA samples: loading buffer = 9: 1. Loaded samples into wells.<br /> | Mixed DNA samples: loading buffer = 9: 1. Loaded samples into wells.<br /> | ||
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<Br> | <Br> | ||
+ | <b>■18th Aug</b><br /> | ||
+ | <Br> | ||
+ | (Construction of Device3)<Br> | ||
+ | ・Electrophoresis<Br> | ||
+ | →Failure: because of deterioration of Loading buffer<Br> | ||
+ | ・Preparation of 0.8 % Agarose Gel<Br> | ||
+ | ・Electrophoresis<Br> | ||
+ | →Failure: because of miss of fdh4AB primer’s sequence<Br> | ||
+ | <b>・Redesign and Order new fdh4AB primer</b> | ||
+ | <Br> | ||
+ | |||
<Br> | <Br> | ||
Latest revision as of 15:06, 26 September 2012
■Experiment
1st week (8.13 - 8.19)
2nd week (8.20 - 8.26)
3rd week (8.27 - 9. 2)
4th week (9. 3 - 9. 9)
5th week (9.10 - 9.16)
6th week (9.17 - 9.23)
7th week (9.24 - 9.30)
Experiment
■1st week (8.13 - 8.19)
■ 14th Aug
(Construction of Device3)
・PCR of vector (BBa_K299009) and insert (fdh4AB)
・Preparation of 1.5 % Agarose Gel Electrophoresis
→Failure: because of Preparation of Gel protocol miss
・Preparation of 1.5 % Agarose Gel Electrophoresis (again)
→Failure: because of Preparation of Gel protocol miss
・Preparation of 1.5 % Agarose Gel
→Failure: because of PCR protocol miss
・PCR of vector (BBa_K299009) and insert (fdh4AB)
・Preparation of 0.8 % Agarose Gel
・Correction of the PCR protocol and Preparation of Gel protocol
■15th Aug
(Construction of Device3)
・Electrophoresis of previous sample
→Failure: because of PCR protocol miss
・PCR of vector (BBa_K299009) and insert (fdh4AB)
・Preparation of 0.8 % Agarose Gel Electrophoresis
→Failure: because of smear
・PCR of vector (BBa_K299009)
・Digestion
1. Mixed following: total 20µL
(DW 15µL / DNA solution 2µL / 10x L buffer 2µL / SacI 1µL;)
2. Incubated at 37 °C for 1 hour.
3. Heat inactivated at 65 °C for 10 minutes.
4. Mixed: Total 50µL
(DW 24µL / Reaction solution 20µL / 10x K buffer 5µL / BamHI 1µL;)
5. Incubated at 37 °C for 1 hour 40 minutes.
・Electrophoresis
Put an agalose gel into the tank, and poured TBE buffer.
Mixed DNA samples: loading buffer = 9: 1. Loaded samples into wells.
Electrophoresed, stopped when samples move to 2/3.
・Gel extraction
Excised the DNA fragment from an agarose gel. For each 100 mg of agarose gel added 200µL Buffer NT.
Incubated sample for 5 – 10 minutes at 50 C. Vortexed the sample briefly every 2 – 3 minuets until the gel slice was completely dissolved.
Placed a NucleoSpin Extract II Columu into a Collection Tube and loaded the sample. Centrifuged for 30 seconds at 11,000x g. Discarded flow-through and placed the column backed into the collection tube.
Added 700µL Buffer NT3 to the NucleoSpin Extact II Column. Centrifuged for 30 s at 11,000x g. Discarded flow-through and placed the column backed into the collection tube.
Centrifuged for 1 min at 11,000x g to remove Buffer NT3 completely.
Placed the NucleoSpin Extract II Column into a new 1.5 ml microcetrifuge tube. Added 30µL Buffer NE and incubated at room temperature for 1 min. Centrifuged for 1 min at 11,000x g.
・Densitometry
Diluted DNA samples 50 times with a solvent.
Turned on the machine; GeneQuant 100.
Poured the solvent 100µL into a cuvette and adjusted 0.
Threw the solvent, poured the DNA sample.
Measured the concentration.
・Results
Concentration of a back bone plasmid was 53 ng /μl.
We could not obtain the target fragments of fdh4AB.
■16th Aug
(Construction of Device3)
・Preparation of 0.8 % Agarose Gel Electrophoresis
→Success: We observed appropriate bands!
・PCR of insert (fdh4AB)
・Check the fdh4AB primer’s sequence
・Refine of PCR products: fdh4AB and Backbone plasmid
Mixed 1 volume of sample with 2 volumes of Buffer NT.
Placed a NucleoSpin Extract II Column into a Collection Tube and loaded the sample. Centrifuged for 30 s at 11,000x g. Discarded flow-through and placed the column back into the collection tube.
Added 700µL Buffer NT3 to the NucleoSpin Extract II Column. Centrifuged for 30 s at 11,000x g. Discarded flow-though and placed the column back into the collection tube.
Centrifuged for 1 minute at 11,000x g to remove Buffer NT3 completely.
Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added 30µL Buffer NE and incubated at room temperature for 1 min. Centrifuged for 1 minute at 11,000x g.
・Densitometry
Diluted DNA samples 50 times with a solvent.
Turned on the machine; GeneQuant 100.
Poured the solvent 100 μl into a cuvette and adjusted 0.
Threw the solvent, poured the DNA sample.
Measured the concentration.
・Results
Concentration of fdh4AB was 210 ng/ μl.
Backbone plasmid No. 01 was 110 ng/ μl
No. 02 was 60 ng/ μl
・Digestion
1. Mixed following
Fdh4AB: total 25µL (milliQ 6.5µL / DNA solution 15µL / 10x L buffer 2.5µL / SacI 1µL)
Backbone plasmid: total 25µL (milliQ 1.5µL / DNA solution 20µL / 10x L buffer 2.5µL / SacI 1µL)
2. Incubated for 1 hour at 37 °C.
3. Heat inactivated at 65 °C for 10 minutes.
4. Mixed following; total 50µL (milliQ 19µL / Reaction solution 25 µL / 10x K buffer 5µL / BamHI 1µL)
5. Incubated at 37 °C for 1 hour.
・Gel extraction
Excised the DNA fragment from an agarose gel. For each 100 mg of agarose gel added 200 μl Buffer NT.
Incubated sample for 5 – 10 minutes at 50 °C. Vortexed the sample briefly every 2 – 3 minuets until the gel slice was completely dissolved.
Placed a NucleoSpin Extract II Columu into a Collection Tube and loaded the sample. Centrifuged for 30 seconds at 11,000 x g. Discarded flow-through and placed the column back into the collection tube.
Added 700 μl Buffer NT3 to the NucleoSpin Extact II Column. Centrifuged for 30 seconds at 11,000 x g. Discarded flow-through and placed the column back into the collection tube.
Centrifuged for 1 minute at 11,000 x g to remove Buffer NT3 completely.
Placed the NucleoSpin Extract II Column into a new 1.5 ml microcentrifuge tube. Added 50 μl Buffer NE and incubated at room temperature for 1 minute. Centrifuged for 1 minute at 11,000 x g.
・Results
We could not obtain the target band of fdh4AB.
・Densitometry
Diluted DNA samples 50 times with a solvent.
Turned on the machine; GeneQuant 100.
Poured the solvent 100 μl into a cuvette and adjusted 0.
Threw the solvent, poured the DNA sample.
Measured the concentration.
・Results
Concentration of backbone plasmid was 48 ng/ μl.
■17th Aug
(Construction of Device3)
・Preparation of 0.8 % Agarose Gel
・Electrophoresis
→Failure: because of different bands
・PCR of insert (fdh4AB)
・Preparation of 0.8 % Agarose Ge
Electrophoresis of PCR products: before digestion of fdh4AB, after PCR products clean-up of fdh4AB, before PCR products clean-up of fdh4AB and diluted 100 times of PCR products of fdh4AB.
Put an agalose gel into the tank, and poured TBE buffer.
Mixed DNA samples: loading buffer = 9: 1. Loaded samples into wells.
Electrophoresed, stopped when samples move to 2/3.
・Results
Every sample was smeared. It seemed that PCR didn’t run well.
■18th Aug
(Construction of Device3)
・Electrophoresis
→Failure: because of deterioration of Loading buffer
・Preparation of 0.8 % Agarose Gel
・Electrophoresis
→Failure: because of miss of fdh4AB primer’s sequence
・Redesign and Order new fdh4AB primer