"
Team:HokkaidoU Japan/Notebook/September29
From 2010.igem.org
(Difference between revisions)
| Line 3: | Line 3: | ||
= Electrophoresis of Yesterday Samples = | = Electrophoresis of Yesterday Samples = | ||
| - | [[Image:HokkaidoU Japan 20100929a.jpg|200px| | + | [[Image:HokkaidoU Japan 20100929a.jpg|200px|center|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]] |
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed. | *electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed. | ||
| Line 83: | Line 83: | ||
*erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI. | *erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI. | ||
| - | [[Image:HokkaidoU Japan 20100929b.jpg|200px| | + | [[Image:HokkaidoU Japan 20100929b.jpg|200px|center|thumb|Electrophoresis of colony PCR sample]] |
| Line 249: | Line 249: | ||
|} | |} | ||
</div> | </div> | ||
| + | |||
| + | *Electrophoresed the samples and pSB1C3 and pSB1T3 which were amplified before.A band of T3SS signal amplified by Quick Taq was observed, but by KOD plus neo wasn't done.Estimated a concentration of T3SS signal and plasmid. | ||
Revision as of 17:24, 27 October 2010
since 13 Jul 2010
since 1 Aug 2010
Electrophoresis of Yesterday Samples
- electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed.
Colony PCR of Arabinose Promoter + RBS + GFP + double terminator
- PCRed 5 colony.
PCR mix
| Reagent | Amount |
|---|---|
| Quick Taq | 25 uL |
| EX-F | 0.5 uL |
| PS-R | 0.5 uL |
| Total | 26 uL |
- PCRed according to the table below.98C and 68C for 35 cycles.
| temp | time |
|---|---|
| 94C | 2 min |
| 94C | 30 sec |
| 68C | 90 sec |
| 4C | hold |
- erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI.
Cultivation of colony
- cultivated the colonies which were used for PCR.Each Samples were cultured in LB contained Ampicillin and 20% Arabinose or contained only Ampicillin.
PCR of T3SS signal again
- We thought colony PCR can't use KOD plus neo.We did a PCR with KOD plus neo again,and also did with Quick Taq.
PCR mix
| Reagent | Amount |
|---|---|
| colony solution | 10 uL |
| DW | 23 uL |
| 10x PCR Buffer | 5 uL |
| 5 mM 4dNTPs | 5 uL |
| 25 mM MgSO4 | 3 uL |
| EX-RBS Primer | 1.5 uL |
| SlrP3 Primer | 1.5 uL |
| KOD plus neo | 1 uL |
| Total | 50 uL |
| Reagent | Amount |
|---|---|
| Quick Taq | 7 uL |
| EX-F | 1.5 uL |
| PS-R | 1.5 uL |
| colony solution | 10 uL |
| Total | 20 uL |
- PCRed according to the table below.98C and 68C for 45 cycles in PCR of KOD. 94C 30 sec and 68C for 40 cycle in PCR of Quick Taq.
| temp | time |
|---|---|
| 94C | 2 min |
| 98C | 10 sec |
| 68C | 60 sec |
| 4C | hold |
| temp | time |
|---|---|
| 94C | 2 min |
| 94C | 30 sec |
| 68C | 90 sec |
| 4C | hold |
- Electrophoresed the samples and pSB1C3 and pSB1T3 which were amplified before.A band of T3SS signal amplified by Quick Taq was observed, but by KOD plus neo wasn't done.Estimated a concentration of T3SS signal and plasmid.
