1.

2.
3.
4.

Make Q21861 RNA (2 hours)

cDNA from RNA for Q21861 (2 hours)
PCR (5 hours)
Digestion of the PCRs made (3 hours)
- Running on Gel (1 hour)
- Chloroform isoamyl Extraction (1 hour)
- Quibit Reading (15 min)
5. Ligation (Overnight)
- Quibit Reading (15 min)
- Making antibiotics stock solution (1 hour)
- Pouring Plates (~20plates) (1 hour)
6. Transformation (4 hours)
- Pick PP2C, NBS-STPK based colonies and grow them in terrific broth (containing
amp+chloramphenicol)
7. Ligation (just one colony for NBS-STPK on a 100-l plate)
8. Making , and MCS transcripts
9. Run Ligation on the Gel
10. Transformation of the ligation made above
11. Pick PP2C, NBS-STPK based colonies and grow them in terrific broth containing
Amp and chloramphenicol
12. Ligation (just one colony for NBS-STPK on a 100 l plate.
13. Making , and MCS transcripts
14. Grow glycerols in LB+Amp Media
15. Run Ligation for NBS-STPK on the Gel (1 hour)
- Quibit Reading (15 minutes)
16. Set up PCR (1 hour)
17. Run PCR on Gels (1 hour)
18. Extract DNA from the , and MCS and DDS34 constructs and the PP2C and NBSSTPK colonies using minipreps (2 hours)
- Quibit Reading for PP2C & NBS-STPK clones for restriction digestion (15 minutes)
19. Set up Digestion reaction
20. Cleaning up , and MCS and DDS34 DNA using P:C:I: extraction (3 hours)
21. Running cleaned DNAs and digested positive PP2C, NBS-STPK clones on GEL.
22. (1 hour)
23. Transformation of the ligation made above (NBS-STPK)
- Quibit Readings (15 minutes)
24. Follow Agilent technologies protocol for induction of target protein (positive
clones for PP2C and NBS-STPK)
- Quibit Readings (15 minutes)
25. Digesting , and MCS and DDS34 DNAs
- Quibit Readings for cleaned , and MCS and DDS34 DNAs (15 minutes)
26. In-Vitro Transcription (m-message m machine protocol)
27. Run , and MCS and DDS34 RNAs on gel.
- Quibit Readings (15 minutes)

1.
2.

Make Q21861 RNA (2 hours)

cDNA from RNA for Q21861 (2 hours)

3.

PCR (4 hours)

Things needed: Template (PP2C), Q2186 cDNA for Rpg5), primers

(PP2CBamHIF/PP2CHindIIIR, NBSEcoRIF/STPKHindIIIR), 5x buffer, Taq, Water,
dNTPs, (MgCl2 not needed)
Amplify PP2C (Regular PCR)
DDS32
1 l
Q21861
5 l
F`
1 l
R`
1 l
5x buffer
5 l
dNTPs
1.5 l
Taq
.2 l
Water
15.3 l
PCR Program:
95 1min
95 1min
62 1min
72 1min
72 5min
Hold 4C

X2
2 l
10 l
2 l
2 l
10 l
3 l
.4 l
30.6 l

Amplify NBS-STPK (Clonetech)

Q21861
F`
R`
Advantage 2 PCR buffer
50x dNTPs
Advantage Taq
Water

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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___________________________________________________________________________ ________________________

4. Digestion of the PCRs made. (20 l reactions) (2 hours)

PP2C Amplicon
BamHI HF
HindIII HF
Cut smart buffer
Water

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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__________________________________________________
__________________________________________________
__________________________________________________
__________________________________________________
__________________________________________________
__________________________________________________
__________________________________________________

Chloroform isoamyl Extraction (1 hour)

___________________________________________________________________________________________________
___________________________________________________________________________________________________
___________________________________________________________________________________________________
___________________________________________________________________________ ________________________

Quibit Reading (15 min)

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__________________________________________________
__________________________________________________
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5. Ligation (Overnight)
__________________________________________________
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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___________________________________________________________________________________________________
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___________________________________________________________________________ ________________________
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___________________________________________________________________________________________________
Quibit Reading (15 min)
__________________________________________________
__________________________________________________
__________________________________________________
__________________________________________________
__________________________________________________
__________________________________________________

6. Making antibiotics stock solution

100mg/ml Amp: Dissolve 1g amp salt in 10ml 100% ethanol
50mg/ml Chloramphenicol: Dissolve .5g chl. salt in 10ml 100% ethanol
34mg/ml Chloramphenicol: Dissolve .34g chl. salt in 10ml 100% ethanol
7. Pouring Plates (~20plates)
500ml total
Things needed: 5g bacto-tryptone, 2.5g yeast-extract, 5g NaCl, 7.5g bacto-agar

Dissolve ingredients in 450ml water

Adjust pH using 0.5M NaOH , bring volume upto 500ml and autoclave
Cool to 55C and add antibiotics.

8. Transformation
Things needed: BL21-DE3 plyse gold cells, 42C water bath, ligation reactions,
pre-warmed SOC media, amp+chloramphenicol plates

Follow Invitrogen one shot cells protocol for transformation

Follow Agilent technologies protocol for induction of target protein

___________________________________________________________________________________________________
___________________________________________________________________________________________________
___________________________________________________________________________________________________
___________________________________________________________________________ ________________________
___________________________________________________________________________________________________
___________________________________________________________________________________________________
___________________________________________________________________________________________________
___________________________________________________________________________________________________
___________________________________________________________________________________________________

9. Pick PP2C, NBS-STPK based colonies and grow them in terrific broth containing
Amp and chloramphenicol
___________________________________________________________________________________________________
___________________________________________________________________________________________________
___________________________________________________________________________________________________
___________________________________________________________________________ ________________________
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

11. Making , and MCS transcripts

Grow glycerols in LB+Amp Media
___________________________________________________________________________________________________
___________________________________________________________________________________________________
___________________________________________________________________________________________________
___________________________________________________________________________ ________________________
12. Run Ligation on the Gel

13. Transformation of the ligation made above

Things needed: BL21-DE3 plyse gold cells, 42C water bath, ligation
reactions, pre-warmed SOC media, amp+chloramphenicol plates
________________________________________________________________________________________________
________________________________________________________________________________________________
________________________________________________________________________________________________
________________________________________________________________________________________________
________________________________________________________________________________________________

14. Pick PP2C, NBS-STPK based colonies and grow them in terrific broth containing
Amp and chloramphenicol
___________________________________________________________________________________________________
___________________________________________________________________________________________________
___________________________________________________________________________________________________
___________________________________________________________________________ ________________________
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
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
17. Run Ligation for NBS-STPK on the Gel (1 hour)
18. Quibit Reading
________________________________________________________________________________________________
________________________________________________________________________________________________
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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________________________________________________________________________________________________
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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
________________________________________________________________________________________________
________________________________________________________________________________________________
________________________________________________________________________________________________
________________________________________________________________________________________________
________________________________________________________________________________________________

22. Quibit Reading for PP2C and NBS-STPK clones for restriction digestion
________________________________________________________________________________________________
________________________________________________________________________________________________
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)
________________________________________________________________________________________________
________________________________________________________________________________________________
________________________________________________________________________________________________
________________________________________________________________________________________________
25. Running cleaned DNAs and digested positive PP2C, NBS-STPK clones on GEL.
(1hour)

26. Transformation of the ligation made above (NBS-STPK)

Things needed: BL21-DE3 plyse gold cells, 42C water bath, ligation reactions,
pre-warmed SOC media, amp+chloramphenicol plates
_____________________________________________________________________________________________
_____________________________________________________________________________________________
_____________________________________________________________________________________________

Follow Invitrogen one shot cells protocol for transformation

Follow Agilent technologies protocol for induction of target protein

27. Quibit Readings

__________________________________________________
__________________________________________________
__________________________________________________
__________________________________________________
28. Follow Agilent technologies protocol for induction of target protein (positive
clones for PP2C and NBS-STPK)
_____________________________________________________________________________________________
_____________________________________________________________________________________________
_____________________________________________________________________________________________
_____________________________________________________________________________________________
_____________________________________________________________________________________________
29. Quibit Readings
__________________________________________________
__________________________________________________
__________________________________________________
__________________________________________________
30. Digesting , and MCS and DDS34 DNAs

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
__________________________________________________
__________________________________________________
__________________________________________________
__________________________________________________
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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___________________________________________________________________________________________________

33. Run , and MCS and DDS34 RNAs on gel.

34. Quibit Readings

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____________________________________________
____________________________________________
____________________________________________
____________________________________________
35.
(25 l reaction)
(2.5+2.5+2.5+17.5FES)

MCS
DDS34
FES

DDS34 Infection (32

Plants)
80
80
80
560

MCS Infection (32 Plants)

80
80
80
560