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Amplification and labelling with fluorescent dyes consists of:

 

Amplification and Fluorescent Labelling of DNA

 

Round A: Two rounds of DNA synthesis using a partially degenerate primer (primer A), T7 sequenase and slow ramp times.

Round A primer sequence (name = T-PCRA) 5'-GTTTCCCAGTCACGATCNNNNNNNNN-3'

 

Round A Setup

 

DNA to be amplified and labelled

7 l

5X buffer

2 l

Primer A (40M)

1 l

Program thermocycler for the following cycle conditions

 

2 min 94 C, hold at 8 C 2 min, then add 5 l rxn mix (while it's at 8 C)*, 8 min ramp to 37 C, 8 min hold at 37 C

 

Repeat once (2 cycles total of the above)

* point to add 2nd aliquot of T7 Sequenase. For 2nd aliquot (5l for 5 reactions), 3.5l sequenase dilution buff + 1.5l T7 Sequenase

 

 

Reaction Mix

 

 

 

Samples

1

3

5

5x buffer

1

3

5

dNTP mix

1.5

4.5

7.5

DTT

0.75

2.25

3.75

BSA

1.5

4.5

7.5

T7 Sequenase

0.3

0.9

1.5

Total

5.05

15.15

25.25

5l aliquots

 

 

 

 

Stocks

 

dNTPs

3mM each

DTT

0.1M

BSA

500g/ml

T7 Sequenase

13U/l

 

T7 sequenase, 5x buffer and sequenase dilution buffer are from is from Amersham, dNTPs are from Pharmacia (Ultrapure, 100mM stocks)

 

After Round A, dilute product to 50l with 35l 1xTE and use 15l in Round B

 

Round B (1): 25 cycles of PCR using a primer (primer B) that anneals to the specific region of primer A, and cold dNTPs.

Round B primer sequence (name = T-PCRB) 5'-GTTTCCCAGTCACGATC-3'


 

Round B Setup

 

 

 

Samples

1

3

5

Template (diluted)

15

---

---

10x PCR buffer

10

30

50

100x dNTPs (diff)

1

3

5

Primer B

2.5

7.5

12.5

Taq

1

3

5

25mM MgCl2*

8

24

40

Water

62.5

187.5

312.5

Total

100

255

425

*[Mg2+] = 2mM final conc.

 

 

 

Add 85l aliquots to each template

 

 

Stocks

 

dNTPs

25mM each

Primer B

500pm/l

Taq (Amplitaq)

5U/l

Final [dNTPs] = 250M

 

 

92 degrees 30 sec

40 degrees 30 sec

50 degrees 30 sec

72 degrees 1 min

25 Cycles

 

After Round B1, use 15l of the product in Round B (fluor)

 

Round B (fluor): 25 cycles of PCR with primer B, and Cy-dUTP. The concentrations of the cold dNTPs and the conditions are different.

Fluorescent Round B Setup

 

 

Samples

1

3

5

Template

15

---

---

10x PCR buffer

5

15

25

F-100x dNTPs (different from above)

0.5

1.5

2.5

Primer B

1

3

5

Cy Dye

3

---

---

Taq

0.5

1.5

2.5

Water

21

63

105

25mM MgCl2

4

12

20

Total

50

96

160

 

 

 

 

Add 32 l aliquots to each template

 

then add Cy Dyes

 

 

 

 

dNTP Stock

 

100mM dATP

10l

100mM dCTP

10l

100mM dGTP

10l

100mM dTTP

5l

1xTE

5l

 

 

92 degrees 30 sec

40 degrees 30 sec

50 degrees 30 sec

72 degrees 2 min

25 Cycles

 

Note: AmpliTaq (Gold), 10X PCR buffer and MgCl2 are from Perkin-Elmer. Cy3-dUTP and Cy5-dUTP are from Amersham

 

Probe purification:

1. Place 450 uL TE (pH 7.4) in Microcon-30 filter. Add reactions to each microcon filter. Centrifuge 7 min. at top speed.

Optional: Repeat TE washes 1-2 times, or until all unincorporated dye is removed.

2. Inspect filters. Centrifuge in 30 sec. intervals until volume is 5-10 uL.

3. Invert filters into fresh tubes. Centrifuge 1 min. to harvest labeled cDNA. Optional: For next day hybridization, store cDNA @ 4C.

4. Mix together Cy3- and Cy5-labeled cDNAs. Concentrate sample to ~10-12 uL using microcon filter or vacuum pump.

5. Add 1 uL polyA DNA or RNA for non-specific hybridization. Add 3 uL 20X SSC for total volume of 12-15 uL.

Optional: Pre-wet millipore filter by adding 5 uL ddH2O. Centrifuge 1 min. at top speed. Remove eluted water with pipet tip.

6. Add probe to filter. (Pipet probe onto filter wall, not directly onto membrane.) Centrifuge 1 min. at top speed.

=> PROBE IS NOW READY FOR HYBRIDIZATION. REMEMBER TO ADD 0.3 uL SDS (10%) TO PROBE as stated in hybridization protocol.


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