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Team:HokkaidoU Japan/Notebook/plastic Week 9
From 2012.igem.org
Contents |
August 27th
Digestion
Digestion to divide BBa_K342001(PhaC) with XbaI and PstI.
And BBa_B0034(RBS) with SpeI and PstI (with three samples).
PhaC (BBa_K342001)
| DNA solution (100 ng/ul) | 12.5 ul |
| XbaI | 1 ul |
| PstI | 1 ul |
| 10xM buffer | 2 ul |
| DW | 3.5 ul |
| Total | 20 ul |
RBS (BBa_B0034)
N0.1
| DNA solution (20.3 ng/ul) | 14.3 ul |
| SpeI | 1 ul |
| PstI | 1 ul |
| 10xH buffer | 2.5 ul |
| DW | 0.2 ul |
| Total | 25 ul |
N0.2
| DNA solution (15.6 ng/ul) | 18.6 ul |
| SpeI | 1 ul |
| PstI | 1 ul |
| 10xH buffer | 2.5 ul |
| DW | 1.9 ul |
| Total | 25 ul |
N0.3
| DNA solution (16.9 ng/ul) | 17.2 ul |
| SpeI | 1 ul |
| PstI | 1 ul |
| 10xH buffer | 2.5 ul |
| DW | 3.3 ul |
| Total | 25 ul |
Gel extraction
Gel extraction for digestion product. Used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
August 28th
Digestion
Digestion to divide PhaA and PhaB with XbaI and PstI.
I digested PhaC(BBa_K342001) with these restriction sites and also XhoI to divide pSB1C3 into pieces, on which PhaC is.
Because the length of pSB1C3 is nearly PhaC.
And I digested PhaC(BBa_K342001) and pSB1C3 with XbaI and SpeI.
PhaA
| DNA solution (125 ng/ul) | 6.6 ul |
| XbaI | 1 ul |
| PstI | 1 ul |
| 10xM buffer | 2 ul |
| DW | 9.4 ul |
| Total | 20 ul |
PhaB
| DNA solution (125 ng/ul) | 4 ul |
| XbaI | 1 ul |
| PstI | 1 ul |
| 10xM buffer | 2 ul |
| DW | 12 ul |
| Total | 20 ul |
PhaC(BBa_K342001)
| DNA solution (125 ng/ul) | 10 ul |
| XbaI | 1 ul |
| PstI | 1 ul |
| XhoI | 5.1 ul |
| 10xM buffer | 2 ul |
| DW | 0.9 ul |
| Total | 20 ul |
Gel extraction
Gel extraction for digestion product. Used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
August 29th
PHB polymer ethanolysis
We did ethanolysis of PHB polymer for 4hrs with sample 2~8
Preparation for GC/MS
We did the preparation for GC/MS with sample 2~8.
PCR
We multiplied pSB1C3 by PCR.
Used two different DNA polymerase, KOD-Plus-Neo and KAPA Taq.
| Solution | Volume(ul) |
| DNA | 1 |
| Suffix-EX | 1 |
| Prefix-PS | 1 |
| MgSO4 | 3 |
| dNTP | 5 |
| 10x KOD-Plus-Neo Buffer | 5 |
| KOD-Plus-Neo | 1 |
| DW | 33 |
| Total | 50 |
| Number | Degree | Second |
| 1 | 94 | 120 |
| 2 | 98 | 10 |
| 3 | 63.7 | 30 |
| 4 | 68 | 60 |
| 5 | 4 | HOLD ↑STEP DOWNに書き換える |
Cycle:2~4 x 35
| Solution | Volume(ul) |
| DNA | 1 |
| Suffix-EX (10uM) | 2 |
| Prefix-PS (10uM) | 2 |
| KAPA Taq | 25 |
| DW | 20 |
| Total | 50 |
| Number | Degree | Second |
| 1 | 95 | 120 |
| 2 | 95 | 30 |
| 3 | 63.7 | 30 |
| 4 | 72 | 180 |
| 5 | 4 | HOLD |
Cycle:2~4 x 35
August 30th
EtOH precipitation
Diegested phaA(X/S) and RBS(S)was performed EtOH precipitation.
Ligation
Colony PCR
August 31th
