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 µl
5X buffer	2 µl
Primer A (40µM)	1 µl

Program thermocycler for the following cycle conditions

 

2 min 94 C, hold at 8 C 2 min, then add 5 µl 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 (5µl for 5 reactions), 3.5µl sequenase dilution buff + 1.5µl 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
5µl aliquots

 

Stocks
dNTPs	3mM each
DTT	0.1M
BSA	500µg/ml
T7 Sequenase	13U/µl

 

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 50µl with 35µl 1xTE and use 15µl  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 85µl aliquots to each template

 

Stocks
dNTPs	25mM each
Primer B	500pm/µl
Taq (Amplitaq)	5U/µl
Final [dNTPs] = 250µM

 

92 degrees       30 sec

40 degrees       30 sec

50 degrees       30 sec

72 degrees       1 min

25 Cycles        

 

After Round B1, use 15µl 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 µl aliquots to each template
then add Cy Dyes

 

dNTP Stock
100mM dATP	10µl
100mM dCTP	10µl
100mM dGTP	10µl
100mM dTTP	5µl
1xTE	5µl

 

 

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.