Team:HokkaidoU Japan/Notebook/September29
From 2010.igem.org
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|style="border-top:1px solid #996"|'''Total''' | |style="border-top:1px solid #996"|'''Total''' | ||
|style="border-top:1px solid #996"|'''20 uL''' | |style="border-top:1px solid #996"|'''20 uL''' | ||
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+ | *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. | ||
+ | |||
+ | <br> | ||
+ | <div style="float:left;"> | ||
+ | {|style="text-align: center" class="protocol" | ||
+ | !temp | ||
+ | !time | ||
+ | |- | ||
+ | |94C | ||
+ | |2 min | ||
+ | |- | ||
+ | |98C | ||
+ | |10 sec | ||
+ | |- | ||
+ | |68C | ||
+ | |60 sec | ||
+ | |- | ||
+ | |4C | ||
+ | |hold | ||
+ | |- | ||
+ | |} | ||
+ | </div> | ||
+ | <div style="float:left;"> | ||
+ | {|style="text-align: center" class="protocol" | ||
+ | !temp | ||
+ | !time | ||
+ | |- | ||
+ | |94C | ||
+ | |2 min | ||
+ | |- | ||
+ | |94C | ||
+ | |30 sec | ||
+ | |- | ||
+ | |68C | ||
+ | |90 sec | ||
+ | |- | ||
+ | |4C | ||
+ | |hold | ||
+ | |- | ||
|} | |} | ||
</div> | </div> |
Revision as of 17:13, 27 October 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 |