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2.
3.
4.
1.
2.
3.
PCR (4 hours)
X2
2 l
10 l
2 l
2 l
10 l
3 l
.4 l
30.6 l
10 l
1 l
1 l
5 l
1 l
1 l
21 l
PCR Program:
95 1min
95 30sec
68 3min
68 3min
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5 l
1 l
1 l
2 l
11 l
NBS-STPK (pellet)
EcoRI
SalI
NEB 3.1
BSA (no need to add)
Water
1 l
1 l
2 l
16 l
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Running on Gel ( 1 hour)
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5. Ligation (Overnight)
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PP2C (digested+cleaned)
Digested Vector
10x Ligase buffer
T4 Ligase
Water
1.4 l
1.5 l
1 l
2 l
5 l
NBS-STPK
Digested Vector
10x Ligase buffer
T4 Ligase
Water
3.3 l
3.3 l
1 l
1 l
1.2 l
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Quibit Reading (15 min)
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8. Transformation
Things needed: BL21-DE3 plyse gold cells, 42C water bath, ligation reactions,
pre-warmed SOC media, amp+chloramphenicol plates
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9. Pick PP2C, NBS-STPK based colonies and grow them in terrific broth containing
Amp and chloramphenicol
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10. Ligation (just one colony for NBS-STPK on a 100 l plate.
NBS-STPK
Digested Vector
10x Ligase buffer
T4 Ligase
Water
6 l
3 l
1 l
1 l
? l
14. Pick PP2C, NBS-STPK based colonies and grow them in terrific broth containing
Amp and chloramphenicol
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15. Ligation (just one colony for NBS-STPK on a 100 l plate.
NBS-STPK
Digested Vector
10x Ligase buffer
T4 Ligase
Water
16. Making , and MCS transcripts
Grow glycerols in LB+Amp Media
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17. Run Ligation for NBS-STPK on the Gel (1 hour)
18. Quibit Reading
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19. Set up PCR
x13
PP2C colony
F`
R`
5x buffer
dNTPs
Taq
Water
PCR Program:
95 1min
95 1min
62 1min
72 1min
72 5min
Hold 4C
NBS-STPK colony
1 l
1 l
5 l
1.5 l
.2 l
14.3 l
13 l
13 l
65 l
19.5 l
2.6 l
185.9 l
F`
R`
Advantage 2 PCR buffer
50x dNTPs
Advantage Taq
Water
1 l
1 l
5 l
1 l
1 l
21 l
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20. Run PCR on Gels (1 hour)
21. Extract DNA from the , and MCS and DDS34 constructs and the PP2C and
NBS-STPK colonies using minipreps (2 hours)
Things needed: 4C centrifuge, Solution 1, Solution 2, Solution 3, isopropanol
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22. Quibit Reading for PP2C and NBS-STPK clones for restriction digestion
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23. Set up Digestion reaction
PP2C clone
1 l
x10
10 l
BamHI HF
HindIII HF
Cut smart buffer
Water
RNAase
1 l
1 l
2 l
14.5 l
.5 l
10 l
10 l
20 l
145 l
5 l
NBS-STPK clone
1 l
EcoRI
SalI
NEB 3.1
Water
RNAase
1 l
1 l
2 l
14.5 l
.5 l
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24. Cleaning up , and MCS and DDS34 DNA using P:C:I: extraction (2hours)
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25. Running cleaned DNAs and digested positive PP2C, NBS-STPK clones on GEL.
(1hour)
39 l
Mlul
1 l
NEB 3.1 2 l
37C
39 l
Spe I
1 l
Cut Smart 2 l
37C
MCS
20 l
BssHII
1 l
NEB 3
2 l
50C
DDS34
20 l
Mlul
1 l
NEB 3.1
2 l
37C
31. Quibit Readings for cleaned , and MCS and DDS34 DNAs
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32. In-Vitro Transcription (m-message m machine protocol)
Things needed: Kit, 37C Incubator, (follow the protocol)
Each rxn.
(65X)
(65X)
MCS (65X)
DDS34(65X)
Water
0.125 l
8.125 l
8.125 l
4.375 l
2x NTPs
1.25 l
81.25 l
43.75 l
10x buffer
0.25 l
16.25 l
8.75 l
Enxyme
0.125 l
8.125 l
4.375 l
Template
80ng
40.625 l
21.875 l
(0.625 of
125ng/l))
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MCS
DDS34
FES