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.
- 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.
- Remove 1x DPBS wash and add 20µl of Sigma Lysis buffer for blood. Repeat for all wells.
- 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.
- Quick spin down the PCR tubes or plate and immediately move to a thermal cycler.
- Incubate at 75ºC for 15 minutes followed by a 4ºC hold.
- 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
- Order and prepare primers for PCR of region of interest.
- PCR reaction and gel electrophoresis.
- 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.
Component Volume(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:
Component Volume(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
Step Temperature Time Denaturation 98ºC 30 seconds 30 cycles 98ºC 10 seconds 60ºC (Tm -5ºC) 30 seconds 72ºC 30 seconds/kb Final extension 72ºC 2 minutesOneTaq * 2x Master Mix Cycling Conditions Step Temperature Time Denaturation 94ºC 30 seconds 30 cycles 94ºC 10 seconds 60ºC (Tm -5ºC) 30 seconds 68ºC 30 seconds/kb Final extension 68ºC 2 minutes
- 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.