Preparation of Single-cell ES Clone Lysate for PCR and Sequencing
Note: Single-cell ES clones are plated individually per well of a 96 well dish and maintained until colonies arise.
  1. Remove media from each well and wash by adding ~100µl of 1x DPBS (2 drops from a graduated squeeze pipet). It is recommended to complete this step for all wells to be lysed before proceeding.
  2. Remove 1x DPBS wash and add 20µl of Sigma Lysis buffer for blood. Repeat for all wells.
  3. Using a P20 pipette set to 20µl and a fresh tip for each well, scratch the bottom of the well in a vertical then horizontal then circular fashion, especially around the corners. Slowly aspirate all of the cell suspension into the tip and expel into a PCR tube or PCR plate well, labeled respective to the particular clone. (clone1 goes into well 1 etc). Seal tubes/plate.
  4. Quick spin down the PCR tubes or plate and immediately move to a thermal cycler.
  5. Incubate at 75ºC for 15 minutes followed by a 4ºC hold.
  6. Carefully remove the seal and add 180µl of Sigma Neutralization buffer to each well. Re-seal and store at 4ºC until ready for use as PCR template.
PCR and Sequencing Protocol
  1. Order and prepare primers for PCR of region of interest.
  2. PCR reaction and gel electrophoresis.
  3. Preparing samples for sequencing.
Preparation of Primers:
Primers can be ordered at 25 nmole scale from any number of companies (IDT, Eton Biosciences etc):
  • Short spin down tubes (10s at max speed)
  • Resuspend oligos with nuclease-free dH2O at 100µM (#nMoles x 10 µl of water).
  • Prepare a working stock mixture of 10µM forward and reverse primers (F+R) as below. Store 100µM primer stocks at -20ºC long term.
    ComponentVolume(µl)
    100µM Forward oligo 20
    100µM Reverse oligo 20
    Nuclease-free water (1X) 160
  • Continue to PCR reaction below or store 10µM F+R at -20ºC until ready for use.
PCR Reaction (25µl):
  • We use Q5 High Fidelity 2x Master Mix (NEB cat# M0492) as well as OneTaq 2x Master mix (NEB cat# M0486).
  • Each 10µM F+R primer mix should be tested with each respective enzyme master mix by amplifying template prepared by the same method you will use in your future experiments.
  • In 200µl PCR ready tubes prepare the reactions for each F+R primer set as below, a master mix can be made to reduce pipetting errors:
    ComponentVolume(µl)
    2X PCR enzyme Master mix 12.5
    10µM F+R primer mix 1.25
    Nuclease-free water (1X) 9.25
    Template DNA <1µg 2.00
  • Place the reactions in a thermal cycler with a heated lid. Cycling conditions for each enzyme shown in tables below:
    Q5 High Fidelity 2x Master Mix Cycling Conditions
    StepTemperatureTime
    Denaturation 98ºC 30 seconds
    30 cycles 98ºC 10 seconds
    60ºC (Tm -5ºC) 30 seconds
    72ºC 30 seconds/kb
    Final extension 72ºC 2 minutes
    OneTaq * 2x Master Mix Cycling Conditions
    StepTemperatureTime
    Denaturation 94ºC 30 seconds
    30 cycles 94ºC 10 seconds
    60ºC (Tm -5ºC) 30 seconds
    68ºC 30 seconds/kb
    Final extension 68ºC 2 minutes
Gel Electrophoresis:
  • Prepare a 1% Agarose gel using 1x TAE buffer to resuspend PCR grade agarose.
  • Weigh Agarose suspension before and after boiling (microwave), add dH2O to make up volume lost during boiling.
  • Add Ethidium Bromide up to 0.5µg/ml (5µl added to 100ml gel suspension) before pouring gel into casting tray with well-comb.
  • After complete solidification, cool gel at 4ºC for 30 minutes before loading for sharper bands.
  • Using 5-10µl of each sample prepare for loading by adding loading dye to 1x concentration and load into gel.(* OneTaq mastermix has loading dye) Use the appropriate mass ladder for your expected base-pair length.
  • Run gel at 100V for 20 minutes before taking the first image.
Sequencing (We use Eton Bioscience sequencing service):
  • Perform PCR cleanup using Exonuclease I Shrimp Alkaline Phosphatase: ExoSAP-IT (Thermofisher cat# 78201.1.ML)
  • Add 1µl of ExoSAP-IT to 9µl of PCR product.
  • Incubate at 37ºC for 30 minutes
  • Add 5µl of ExoSAP-IT treated sample to new 200µl PCR tube and dilute up to 16µl, label and submit with sequencing primer for sequencing according the sequencing service instructions.