molecular tricks @ NovoHelix
General considerations for drop dialysis of CRISPR repair template DNA (ssDNA or dsDNA)
drop dialysis volume. The dialysate should be around 200x – 500x larger than the sample volume. I have dialyzed against
- 18.2 megaohms resistant water (type I water),
- microinjection buffer [10 mM Tris, pH 7.5; 0.1 mM EDTA], or
- BAC microinjection buffer [10 mM Tris, pH 7.5; 100 mM NaCl, 0.1 mM EDTA] depending on the application.
drop dialysis time. For most DNA desalting applications prior to embryo manipulation, the bulk of the salt in the sample is dialyzed within 2 hours. The sample ‘saltiness’ can be measured by a conductivity probe. I invested in a microscale conductivity probe because I prepare pre-implantation embryo media and human pluripotent stem cell media; for batch testing, I use the conductivity and osmometer readings to test lots prior to culture and manipulation. While you can let the dialysis proceed overnight to reduce low-concentration impurities, or alternatively, change the buffer after 2 hours with new buffer and proceed another 2 hours.
stirring. An autoclaved small white PTFE stir bar can be used to reduce drop dialysis time. Place the stir bar on low in the center of a petri dish and stir. While this stirring is generally unnecessary, in a time-crunch prior to an afternoon microinjection session, I have done it without issue. Conductivity/’saltiness’ dropped approximately 25% faster over the course of 2 hours.
DNA size. Let’s consider the DNA size in terms of DNA mass and DNA length because the membrane filters often use a molecular weight cut-off (MWCO) or a pore size (micron) dimension that reflects either mass or length, respectively.
Dickerson et al, 1982 provide DNA length measures.
- 1 bp ≈ 0.33 nm
- 1,000 bp = 1 kb ≈ 0.33 µm
Dickerson RE, Drew HR, Conner BN, Wing RM, Fratini AV, Kopka ML. The anatomy of A-, B-, and Z-DNA. Science. 1982 Apr 30;216(4545):475-85. doi: 10.1126/science.7071593. PMID: 7071593
In general, select membrane pore that is to be retained.
- DNA length. 1 kb = 0.33 µm/0.05 µm porosity = 6.6x bigger.
- DNA mass. The average weight of a base pair (bp) is 650 daltons, by extension 1 kb = 650 kDa. For a 1 kb repair template, the selection of a membrane with less than ~200 kDa MWCO is sufficient. The size of the DNA molecule is inversely related to dialysis equilibrium, and larger pores will reduce dialysis equilibrium timescales.
Membrane chemistry and size. Common dialysis filter membranes used/recommended for embryo manipulation include Millipore/Sigma Cat Nr. VMWP02500 with chemistry mixed cellulose esters (MCE), diameter 25 mm, and porosity 0.05 µm.
DNA volume: membrane diameter recommendations:
- DNA volume less than 25 ul à use 13 mm diameter (Cat Nr. VMWP01300/porosity 0.05 µm or VSWP01300/porosity 0.025 µm)
- DNA volume less than 200 ul à use 25 mm diameter (Cat Nr. VMWP02500)
- DNA volume less than 1 ml à use 45 mm diameter (Cat Nr. VMWP04700)
Tip: For those of us that have big clumsy hands, use sterile forceps to assist in placing the filter shiny side up on the dialysate buffer.
Q: Can I substitute the common protein-nonbinding polyethersulfone/PES filter membrane with porosity 0.05 µm with a mixed cellulose esters (MCE) filter membrane for drop dialysis?
A: Not recommended. I have empirically tested and DNA drop spreads out by capillary action and makes it difficult to recover after a couple of hours of drop dialysis. A slide-a-lyzer dialysis unit with a PES membrane with appropriate porosity should work acceptably.
Consumables. Ensure that all consumables are RNAse-free and non-toxic to the embryo.