This protocol will allow you to quantitatively determine the number of deleted E2A alleles generated from Cre mediated recombination of the E2A^flox allele.  This is particularly useful for accurate analysis of E2A deletion efficiency in small populations of cells where there is not sufficient DNA to perform Southern analysis.

The following protocol is optimized to work with a Roche Light Cycler but it can be modified to work with any quantitative PCR platform.

Sample Preparation

For < 1×10⁶ cells:

• Spin down cells (can be in growth media or PBS) and remove all sup. but 20ul
• Add 1:1 vol:vol of Triton lysis buffer ( 960ul Tris-HCl (pH 7.5) + 20ul 10% Triton X-100 + 20ul 10mg/ml Proteinase K)
• Incubate at 55^oC for 30 min followed by 95^oC for 10 min (PCR program YZ3) – Store at 4^oC
• Quantitative PCR (qPCR) reaction for E2A deletion:2ul           Roche Master Mix (Roche FastStart DNA master SYBR-Green PCR kit)1.6ul        25mM MgCl21ul           10uM YZ-1981ul           10uM E2Aflox for12.4ul      H2O

  2ul           genomic DNA (prepared above)

• Control qPCR reaction for normalization of samples (CD14 locus)2ul           Roche Master Mix (Roche FastStart DNA master SYBR-Green PCR kit)1.6ul        25mM MgCl21ul           10uM CD14 for1ul           10uM CD14 rev

  12.4ul      H2O

  2ul           genomic DNA (prepared above)

• Run all samples together in Roche Light Cycler1) 95^oC 5min2) 95^oC 5sec3) 60^oC 10sec4) 72^oC 1.5min

  5) goto #2 45x

  6) Run melting curve

PCR primers:

CD14 for      5′-GCT CAA ACT TTC AGA ATC TAC CGA C-3′

CD14 rev     5′-AGT CAG TTC GTG GAG GCC GGA AAT C-3′

YZ-198        5′-CGG ATC CAT CCT CGT CTT CAT TGG TAC TG-3′

E2Aflox for   5′-CTG CAC TCC GAA TTG TGC CTG-3′

 

Standard Curve generation and evaluation of PCR reaction sensitivity:

In order to determine the relationship between qPCR values and % E2A deletion in a population cells a standard curve was generated by mixing known numbers of E2A^flox/flox cells with E2A^del/del cells

Standard Curve:

Each point was made from 1×10⁵ total E2A^flox + E2A^del cells mixed in the following ratios:

10% del : 90% flox

25% del : 75% flox

50% del : 50% flox

75% del : 25% flox

90% del : 10% flox

Genomic DNA was prepared & qPCR was performed as described above

Samples were normalized to CD14 & the E2A^del qPCR values were plotted over % E2A deletion

 

Fig. 1: Example of a standard curve for the detection of E2A deletion in a mixed population of alleles

 

Samples with unknown % E2A deletion are normalized to CD14 and % E2A deletion is determined by plotting the E2A^del qPCR value on the Standard Curve

This PCR reaction can reliably amplify as few as 40 copies of E2A^del allele (the 10% del : 90% flox sample)

 

Additional note: Excessive numbers of dead cells can significantly influence this protocol. This is particularly important when working with cultured cells (Abelson pre-B cell lines).  In general, make sure that there is < 5% dead cells in the population in order to obtain reliable, quantitative results.

 

 

Immunology Donkey endorses this protocol

Back to genotyping