TeSLA for TL measurement

Oligos for telomeric DNA ligation 

Oligos for Subtelomeric  DNA ligation

• TeSLA ADR1 C3S has C3 spacer at 3’ end (avoid DNA ligation)
• TeSLALA P22 TA and TeSLA P22 AT have C3 spacer at 3’ end (avoid DNA ligation) and phosphorylation at 5’ end (facilitate DNA ligation)
• pre-anneal TeSLA ADR1 C3S with TeSLA P22 TA and TeSLA ADR1 C3S with TeSLA P22 AT to make stocks for subtelomeric DNA ligation

1. 40 µl TeSLA ADR1 C3S (100 µM) + 40 µl TeSLA p22 TA (100 µM) + 20 µl 5X STE buffer (40 µM TA adapter)
2. 40 µl TeSLA ADR1 C3S (100 µM) + 40 µl TeSLA p22 AT (100 µM) + 20 µl 5X STE buffer (40 µM AT adapter)
3. Heat TA adapter and AT adapter up to 94 - 100℃ and gradually cool them down to room temp to make 40 µM stocks. For this step, I usually put samples in beaker with boiling water on heat plate for 5 min and then turn the heat plate off to let samples slowly cool down to room temp.

    5X STE: 50 mM Tris pH 8.0, 250 mM NaCl, 5 mM EDTA

Oligos for TeSLA PCR

Day 1

35℃ overnight

10X CutSmart Buffer is from New England Biolabs (NEB)

10 mM ATP from NEB (cat# P0756S)

Telo 1-6: mix Telo 1, 2, 3, 4, 5, and 6 together to make 10^-2 µM for each

T4 ligase is from NEB (2000000 units/ml cat# M0202M)

Day 2

1. 65℃ 10 min to inactivate reactions from day 1

2. 

    25℃ 2hr

    CviAII is from NEB (10000units/ml, cat# R0640S) 

3.

    37℃ 2hr

    BfaI (10000 units/ml, cat# R0568S), MseI (10000 units/ml, cat# R0525S) and NdeI (20000 units/ml, cat# R0111S) are from NEB

4. 

    37℃ 1hr -> 80℃ 20 min and then gradually reduce temp to be RT

    rSAP (1000 units/ml, cat# M0371S) is from NEB

5. 

    16℃ overnight

Day 3

1. 65℃ 10 min to Inactivate reactions from Day2 step 5
2. Dilute reaction from step 1 to be 10 pg DNA/ µl (50X dilution)
3. PCR

Program

FailSafe polymerase and 2X PreMix H are from epicenter, cat# FS9901K and choose FSP995H-INCL for PreMix H

After PCR, run 0.85% gel and then perform Southern blotting using telomere probe.