Mouse Blastocyst DNA Extraction for PCR Amplification


  • Blastocysts placed in 10 ul of water in PCR tubes and frozen at -20C or -80C
  • 2X Blastocyst Lysis Buffer
  • Pipetman
  • RNAse Free Pipet Tips
  • PCR tubes
  • PCR primers for the gene of interest
  • Gene specific PCR primers and Taq polymerase of choice
  • Thermal Cycler for pCR
  • 1 M KCl (Sigma Cat. no. 60142)
  • Tween 20 (Sigma Cat. no. P9416)
  • 2% Gelatin (Sigma Cat. no. G1393)
  • Nuclease Free Water (Sigma Cat. no. W4502)
  • 10 mg/ul yeast tRNA (Sigma Cat. no. R5636) store at -20C
  • 20 mg/ml Proteinase K (New England BioLabs Cat. no. P8107S) store at -20C



Blastocyst Lysis Buffer Base (100 mM Tris-HCl, pH 8.3 100 mM KCl, 0.02% gelatin, 0.45% Tween 20)

For 10 ml, mix together:

  • 2.0 ml 1 M Tris-HCl (pH 8.3)
  • 2.0 ml 1 M KCl
  • 40 ul 2% Gelatin
  • 90 ul Tween 20
  • 5.87 ml Water

Store at room temperature.


2X Blastocyst Lysis Buffer (add 60ug/ml yeast tRNA, 125 ug/ml Proteinase K)

For 1 ml, mix together:

  • 1 ml Blastocyst Lysis Buffer Base
  • 16 ul 10 mg/ul Yeast tRNA
  • 62. ul 20 mg/ml Proeinase K


Blastocyst Lysis to produce crude DNA solution

Add 10 ul of 2X Blastocyst Lysis Buffer to each blastocyst frozen in a PCR tube

Place PCR tubes in the thermal cycler

Run the following program to prepare the crude DNA solution:

56C for 10 min
95C for 10 min
4C indefinitely

Store crude lysate at -20C


Blastocyst PCR

Use 4 ul of crude blastocyst DNA in a 20 ul PCR reaction to detect the gene of interest.

nota bene; We have had good results with KOD polymerase when other Taq polymerases didn’t amplify.

Protocol adapted from Sakurai T, Watanabe S, Kamiyoshi A, Sato M, Shindo T. 2014. A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice. BMC Biotechnol. 14:69. PMID: 25042988



Highly specific and sensitive assays should be used because most PCR reactions are based on the use of 1 ng DNA per PCR reaction. One blastocyst is about 60 cells; a single cell contains about 6 pg of DNA; thus the starting material for a blastocyst PCR is 4 ul crude lysate X (60 cells X 6 pg DNA/cell)/20 ul crude lysate) or 72 pg DNA per PCR reaction.


Primer Design Suggestions for Specific and Sensitive PCR Assays

Use Primer-Blast to pick primers:

Adjust Primer Parameter default settings

  • Minimum primer melting temperature: change to 60°C
  • Optimal primer melting temperature: change to 63°C
  • Maximum primer melting temperature: change to 66°C Minimum primer melting temperature difference: change to 1°C

Adjust Specificity Checking Parameters

  • Click box to turn on “Enable search for primer pairs specific to the intended PCR template” Set Search Mode to “Automatic”
  • Set Database to “Genome (reference assembly from selected organisms)”
  • Set Organism to “Mus musculus (taxid: 10090)”

Click on “Advanced Parameters”

  • Set Primer Size Min to 27
  • Set Primer Size Opt to 29
  • Set Primer Size Max to 31

Stratman et al. reported primers of 27-30 nucleotides made up of 50-60% GC content and that will produce a 100-500 bp PCR product uniformly detect genomic templates with single copy sensitivity.

Stratman JL, Barnes WM, Simon TC. 2003. Universal PCR genotyping assay that achieves single copy sensitivity with any primer pair. Transgenic Res. 12:521-522. PMID: 12885173


PCR Enhancer Mix

The 5X CES PCR enhancer mix described by Ralser et al. can improve the results obtained from difficult PCR reactions. The authors further recommend that the PCR reaction buffer contain the following final concentrations of components: 65 mM Tris–HCl, 16.6 mM (NH4)2SO4, 3.1 mM MgCl2, and 0.01% (v/v) Tween 20 at a pH of 8.8.

2.7 M betaine 6.7 mM DTT 6.7% DMSO 55 ug/ml BSA

Ralser M, Querfurth R, Warnatz HJ, Lehrach H, Yaspo ML, Krobitsch S. 2006. An efficient and economic enhancer mix for PCR. Biochem Biophys Res Commun. 347:747-751. PMID: 16842759

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