Researchers around the world are using our gBlocks® Gene Fragments successfully for a wide range of applications. We frequently receive questions about the best way to handle these fragments, so we would like to share a few tips from the scientists in our synthetic biology group.
Resuspending gBlocks Gene Fragments
gBlocks Gene Fragments ordered in tubes are provided dry and must be resuspended. (Note: gBlocks Gene Fragments ordered in plates come in 25 µL of nuclease-free water at 10 ng/µL.) We have evidence of these dsDNAs occasionally adhering to the plastic tubes, and therefore, strongly recommend the 50°C incubation described below in step 4. Here is our recommended protocol for resuspension:
- Before opening the tube, spin it down in a microcentrifuge for 3–5 seconds to ensure the DNA is in the bottom of the tube. The pellet can become statically charged and, without this step, can either fly out of the tube or remain in the cap, resulting in loss of yield.
- Add molecular grade water, or a buffer such as IDTE, to reach a final concentration of 10 ng/µL. Our experiments have shown that storage concentrations <1 ng/µL result in loss of material due to adherence to the plastic tube in the absence of a carrier such as tRNA.
- Vortex briefly.
- Incubate at approximately 50°C for 15–20 min. Heating the tube will ensure the solvent comes in contact with the tiny pellet, even if it is stuck to the side of the tube. Thus, this step will increase the likelihood that the entire pellet will be resuspended.
- Briefly vortex and centrifuge.
- Verify the final concentration (see below).
See the DECODED article, Tips for resuspending and diluting your oligonucleotides, for more advice on nucleic acid handling.
Quantifying gBlocks Gene Fragments
gBlocks Gene Fragments are delivered in amounts of 250–1000 ng. For quantification, we recommend using methods designed for small sample volumes, such as those using the NanoDrop™ (Thermo Fisher Scientific) or Qubit® instruments (Thermo Fisher Scientific). Note that when comparing resulting concentration values across instruments, variances may be seen due to the methods employed by each instrument. Ensure accurate measurements by performing the following steps:
- Take the measurement as soon as the sample is prepared for the instrument.
- Repeat quantification of each sample twice.
- When using a Nanodrop instrument, test the sample resuspension solution alone between each sample measured. This ensures that there is nothing in the water or buffer that absorbs at A260 and would artificially inflate your sample readings.
Calculating copy number
It is often necessary to dilute the resuspended gBlocks Gene Fragment to a specific copy number/µL. The molecular weight and fmol/ng conversions for each gBlocks fragment are provided on the spec sheet provided with the fragment (Figure 1).
Figure 1. Specification sheet information needed for copy number calculation.
Calculating copy number from concentration measurements
You can easily convert your concentration from ng/µL to copy number/µL by using the formula below.
(C) (M) (1 x 10–15 mol/fmol) (Avogadro’s number) = copy number/µL
Where C is the current concentration of the gBlocks Gene Fragment in ng/µL, and M is the molecular weight in fmol/ng, as provided on the spec sheet.
Example: A gBlocks Gene Fragment, with the properties shown in Figure 1 (M= 2.12 fmol/ng), is resuspended to 10 ng/µL:
(10 ng/µL) (2.12 fmol/ng) (1 x 10–15 mol/fmol) (6.022 x 1023) = 1.28 x 1010 copies/µL
Have you thought about these gBlocks fragment applications?
gBlocks Gene Fragments have been used in a wide range of applications including CRISPR-mediated genome editing, antibody research, codon optimization, mutagenesis, and aptamer expression. They can also be used for generating qPCR standards. With gBlocks Gene Fragments, IDT provides a tool that accelerates your research and makes synthetic biology easier and more accessible than ever. Learn more about gBlocks Gene Fragments at www.idtdna.com/gBlocks.