Corning Ultra-Low Attachment Dishes (Fisher Scientific, cat. no. 05-539-100)
0.2 ml PCR tube strips (Eppendorf, cat. no. 951010022)
1.5 ml tubes, DNase and RNase-free (Eppendorf, cat. no. 022431021)
2 ml tube, DNase and RNase-free (Eppendorf, cat. no. 022431048)
Falcon 15mL tubes (Fishersci, cat. no. 14-959-49B)
ART Barrier Pipette tips 10 µL (Fisher Scientific, cat. no. 21-402-482)
ART Barrier Pipette tips 100 µL (Fisher Scientific, cat. no. 21-402-485)
ART Barrier Pipette tips 200 µL (Fisher Scientific, cat. no. 21-402-486)
ART Barrier Pipette tips 1000 µL (Fisher Scientific, cat. no. 21-403-03)
ΔCRITICAL Same quality tips can be replaced, but all pipette tips should be sterile, DNase- and RNase-free with low retention and filter.
Qubit 2.0 Fluorometer (Invitrogen, cat. no. Q32866)
Qubit Assay Tubes (Invitrogen, cat. no. Q32856)
DynaMag-96 Side Magnet (Life technologies, cat. no. 12331D)
Microseal 'B' Adhesive Seals (Bio-Rad, cat. no. MSB-1001)
Multiplate Low-Profile 96-Well Unskirted PCR Plates (Bio-rad, cat. no. MLL-9601)
The STRIPPER Micropipetter (Origio, cat. no. MXL3-STR)
Tips - 75μm (20/pk) (Origio, cat. no. MXL3-75)
Vortexer (VWR, cat. no. 58816-121)
Mini Centrifuge (Fisher Scientific, cat. no. S67601APRMO)
CFX96 Touch Real-Time PSCR Detection System (Bio-Rad, cat. no. 185-5195)
CFX Manager Software (Bio-Rad, version 3.1)
Thermal Cycler (Eppendorf, Mastercycler gradient)
NanoDrop Lite Spectrophotometer (Thermo Scientific, cat. no. NDNDLUSCAN)
Dissecting microscope (Nikon, cat. no. SMZ-2B)
PCR workplace (LABCONCO, purifier class II biosafety cabinet)
Aliquot water. Nuclease-free water is suggested to aliquot into 1.5 ml Eppendorf tube (1 ml/tube) to avoid contamination in PCR workplace. Dispose of the water tube after each use. In this protocol all the water is nuclease-free and sterile.
Single cell lysis buffer (LB). Mix the following components and make 1 ml of lysis buffer in 1.5 ml tube. Aliquot LB at 100 µl/tube and store at 4°C or colder. SEE TABLE 1
Primer pair mix. Centrifuge primer powders in caped tubes at 14000 rpm for 5 min prior to adding water. Make a 100 µM of stock solution for each primer with water, and then aliquot 10 µl of each stock solution into 1.5 ml Eppendorf tube respectively. Add 90 µl of water into each tube and make 10 µM of working solution of primers. Combine the forward and reverse primers of working solution for telomere and Alu respectively to make a primer pair mix with final 200 µl volume and 10 µM concentration. Label the primer pair mix tube with Tel-F/R and Alu-F/R respectively, then mark 10 µM. Primer stock solution can be stored at -20°C or colder, and primer pair mixes can be store at 4°C for up to 2 weeks or at -20°C for long term.
ΔCRITICAL Mixing primer pair reduces the possibility of contamination.
gDNA extraction Extract DNA from cell pellets of human fibroblast and embryonic stem cell (hESC) respectively using Quick-gDNA Mini Prep kit and elute DNA with water. Quality of eluted DNA is checked by using Nano-drop Lite, and the concentration is measured by Qubit 2.0 fluorometer. Then aliquot the DNA and store at -20°C.
ΔCRITICAL High quality of eluted DNA is indicated by ratio 1.8-1.9 of A280/A260.
Working solution of Positive Template Controls (7 pg/µl and 5 ng/µl of hESC gDNA) Mix the stock of hESC gDNA by vortex thoroughly and spin shortly. In PCR workplace, aliquot hESC gDNA into 1.5 ml Eppendorf tube and make 500 µl of working solution for 2ndPTC (5 ng/µl) by adding water, mix by vortex and spin shortly. To make 1stPTC (7 pg/µl), mix 500 µl of water and 0.7 µl of hESC gDNA (5 ng/µl) in 1.5 ml Eppendorf tube, mix well and spin shortly. Both of positive template controls working solution can be stored at -20°C for long term.
□ PAUSE POINT Positive controls can be replaced with different genomic DNA and is not limited on hESC gDNA.