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Team:HokkaidoU Japan/Notebook/September24
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*Follow quality check | *Follow quality check | ||
| - | + | = Miniprep of Arac+RBS+pSB1A3 = | |
#Transfered E.coli solution to 1.5 mL tubes, 1 mL each | #Transfered E.coli solution to 1.5 mL tubes, 1 mL each | ||
| - | #Centrifuged at 4C, | + | #Centrifuged at 4C, 15000 rpm for 1 min |
#Discarded the supernatant | #Discarded the supernatant | ||
#Suspended on 125 uL of Buffer P1 each | #Suspended on 125 uL of Buffer P1 each | ||
#Added 175 uL Buffer N3 each mixed by inversion | #Added 175 uL Buffer N3 each mixed by inversion | ||
| - | #Centrifuged at 4C, | + | #Centrifuged at 4C, 13000 rpm for 10 min |
#Transfered the supernatant to filtration column | #Transfered the supernatant to filtration column | ||
| - | #Centrifuged at 4C, | + | #Centrifuged at 4C, 13000 rpm for 1 min |
#Discarded the flow-through | #Discarded the flow-through | ||
#added 500 uL of Buffer PB to filtration column | #added 500 uL of Buffer PB to filtration column | ||
| - | #Centrifuged at 4C, | + | #Centrifuged at 4C, 13000 rpm for 1 min |
#Discarded the flow-through centrifuged for 1min to remove remaining buffer | #Discarded the flow-through centrifuged for 1min to remove remaining buffer | ||
#Transfered filtration column to a new 1.5 ml tube | #Transfered filtration column to a new 1.5 ml tube | ||
#Resuspended on 50 ul of TE and incubated at RT for 1min | #Resuspended on 50 ul of TE and incubated at RT for 1min | ||
| - | #Centrifuged at 4C, | + | #Centrifuged at 4C, 13000 rpm for 1 min |
| - | == | + | == Electrophoresis of minipreped samples == |
| - | + | #Mixed 1 uL of sample with loading buffer 1 uL | |
| + | #Added 6 uL of λ/Hind Ⅲ EcoR Marker | ||
| + | #Electrophoresed | ||
| - | [[Image:HokkaidoU Japan 20100924a.JPG|200px|left|thumb|]] | + | [[Image:HokkaidoU Japan 20100924a.JPG|200px|left|thumb|Electrophoresis of minipreped samples]] |
| + | Compared to marker plasmid is about 7000 bp long | ||
| - | + | Arabinose Promoter+RBS+pSB1A3 is 3455 bp long, so it might be tandem | |
| - | + | From the picture estimate of concentration was about 30 ng/uL | |
| - | + | ||
| - | + | ||
<br><br><br><br><br> | <br><br><br><br><br> | ||
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| + | = Digestion of plasmid and GFP+double terminator = | ||
| + | Digestion mix According to the table below | ||
| + | |||
{|style="text-align: center ;float:left;" class="protocol" | {|style="text-align: center ;float:left;" class="protocol" | ||
!Reagent | !Reagent | ||
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|style="border-top:1px solid #996"|'''50 uL''' | |style="border-top:1px solid #996"|'''50 uL''' | ||
|} | |} | ||
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<br><br><br><br><br><br><br><br><br><br><br><br> | <br><br><br><br><br><br><br><br><br><br><br><br> | ||
| - | #37C | + | #incubated at 37C for 60 min |
| - | #Mycrocon YM- | + | #purified samples with Mycrocon YM-10 |
| - | #4C, | + | #sampleが500 uLになるようにTEを加え、カラムに移した。 |
| + | #centrifuged at 4C,14000 G for 1h | ||
#上澄みが30 uL前後残ったので、それを新しいcollection tubeに移し、カラムを逆さまにして挿入した。 | #上澄みが30 uL前後残ったので、それを新しいcollection tubeに移し、カラムを逆さまにして挿入した。 | ||
| - | #4C, | + | #centrifuged at 4C,1000 G for 3min |
#GFP+double terminatorが30 uL、plasmid25 uL回収できたので、各1/10量の3M CH3COONaを加えた。 | #GFP+double terminatorが30 uL、plasmid25 uL回収できたので、各1/10量の3M CH3COONaを加えた。 | ||
#100%EtOHを回収量の2.5倍量加え、voltexにかけた。 | #100%EtOHを回収量の2.5倍量加え、voltexにかけた。 | ||
| - | #4C, | + | #centrifuged at 4C,15000 rpm for 10min |
#上澄みを捨て、70%EtOHを500 uL加え、voltexにかけた。 | #上澄みを捨て、70%EtOHを500 uL加え、voltexにかけた。 | ||
| - | #4C,15000rpm | + | #centrifuged at 4C,15000rpm for 5min |
#上澄みを捨て、真空装置へ入れよく乾燥させた。 | #上澄みを捨て、真空装置へ入れよく乾燥させた。 | ||
#2 uLのTEで溶かした。 | #2 uLのTEで溶かした。 | ||
Latest revision as of 16:19, 27 October 2010
since 13 Jul 2010
since 1 Aug 2010
- Miniprep of Arac+RBS+pSB1A3
- Follow quality check
Miniprep of Arac+RBS+pSB1A3
- Transfered E.coli solution to 1.5 mL tubes, 1 mL each
- Centrifuged at 4C, 15000 rpm for 1 min
- Discarded the supernatant
- Suspended on 125 uL of Buffer P1 each
- Added 175 uL Buffer N3 each mixed by inversion
- Centrifuged at 4C, 13000 rpm for 10 min
- Transfered the supernatant to filtration column
- Centrifuged at 4C, 13000 rpm for 1 min
- Discarded the flow-through
- added 500 uL of Buffer PB to filtration column
- Centrifuged at 4C, 13000 rpm for 1 min
- Discarded the flow-through centrifuged for 1min to remove remaining buffer
- Transfered filtration column to a new 1.5 ml tube
- Resuspended on 50 ul of TE and incubated at RT for 1min
- Centrifuged at 4C, 13000 rpm for 1 min
Electrophoresis of minipreped samples
- Mixed 1 uL of sample with loading buffer 1 uL
- Added 6 uL of λ/Hind Ⅲ EcoR Marker
- Electrophoresed
Compared to marker plasmid is about 7000 bp long
Arabinose Promoter+RBS+pSB1A3 is 3455 bp long, so it might be tandem
From the picture estimate of concentration was about 30 ng/uL
Digestion of plasmid and GFP+double terminator
Digestion mix According to the table below
| Reagent | Amount |
|---|---|
| DW | 5.6 uL |
| 10x H Buffer | 1 uL |
| 0.1% BSA | 1 uL |
| Spe I | 0.2 uL |
| Pst I | 0.2 uL |
| plasmid | 2 uL |
| Total | 10 uL |
| Reagent | Amount |
|---|---|
| DW | 34 uL |
| 10x M Buffer | 5 uL |
| 0.1% BSA | 5 uL |
| Xba I | 4 uL |
| Pst I | 0.4 uL |
| GFP+double terminator | 1.6 uL |
| Total | 50 uL |
- incubated at 37C for 60 min
- purified samples with Mycrocon YM-10
- sampleが500 uLになるようにTEを加え、カラムに移した。
- centrifuged at 4C,14000 G for 1h
- 上澄みが30 uL前後残ったので、それを新しいcollection tubeに移し、カラムを逆さまにして挿入した。
- centrifuged at 4C,1000 G for 3min
- GFP+double terminatorが30 uL、plasmid25 uL回収できたので、各1/10量の3M CH3COONaを加えた。
- 100%EtOHを回収量の2.5倍量加え、voltexにかけた。
- centrifuged at 4C,15000 rpm for 10min
- 上澄みを捨て、70%EtOHを500 uL加え、voltexにかけた。
- centrifuged at 4C,15000rpm for 5min
- 上澄みを捨て、真空装置へ入れよく乾燥させた。
- 2 uLのTEで溶かした。
- -20Cで凍結保存した。
Transformation of BAC Vector
- Used DH5Alpha and MG1655 strains for electroporation
- Plated at 19:15
- Will be incubated for 18 h
