Universal-STELA
Tsung-Po Lai
 Day 1: DNA digestion
Digest 200ng of genomic DNA using NEB enzymes in 50μl final.

MseI

NdeI

NEBuffer 2 + BSA
Overmight at 37°C

Day 2: 65℃ for 20 min to inactivate restriction enzymes

Day 2: Ligation - Panhandle
Use the 0.5 μl LoBind Tubes

Digested DNA
10ng

NEBuffer2 (10X)
2μl

ATP (10mM)
2μl

Annealed panhandle* (40µM)
2.5μl (100 pmol final)

T4 DNA Ligase
0.5μl (20 units)

ddH2O

16°C Overnight

to total 20μl
Day 3: Ligation - Telorette

C-Telorette mix (10-2µM each)
2.5μl

ATP (10mM)
1.5μl

NEBuffer 2 (10X)
0.5 μl

T4 DNA ligase
0.5μl (20 units)

35°C Overnight
Inactivate ligase 65°C for 20min
Store at 4°C
Final volume is now 25 μl

Day 4: PCRs
Samples are now at 400 pg/μl, in order to get clear discrete band, representative of single telomere length,
one needs to perform dilution series.
Prepare 3 to 7 points dilution: 40 ; 30; 20; 10 and 5 pg/μl 10 pg/μl is usually the best for Fibroblast;

5 pg/μl for HeLa
For PCR, choice of Mix depends on ability of the enzyme to hold long extension (15min) over 25 cycles.
Fail Safe (12 μl mix) works just fine.
PCR profile
94°C
X25
2min
94°C
15sec
58°C
30sec
72°C
15min
4°C
Hold
(12min for HeLa)

Run PCR products on a 0.85% Agarose Gel; use TRF ladder (0.2 ng)

Denature the Gel for 30min
1.5M NaCl, 0.5M NaOH
Neutralize for 30min

1.5M NaCl, 0.5M Tris HCl pH 8.0

Wash with 10xSSC for 5-10 min

TRANSFER
Hybridization
Transfer into hybridization tube – use TP’s bench – as he doesn’t use Radioactivity.

Pre-Hyb
Add 10ml of DIG Easy Hybridization (Roche #11603558001) Version II.
42°C

30min
Hyb
Add 10ml of fresh DIG Easy Hyb completed with 1-2nM probe (10 μl of a 1-2μM solution).
42°C
Overnight