Researchers around the world are using our eBlocks™, gBlocks™ and gBlocks HiFi Gene Fragments successfully for a wide range of applications. This article shares a few tips and tricks about handling these gene fragments from IDT’s team of synthetic biology experts.
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. Storage concentrations <1 ng/µL may result in loss of material due to adherence to the plastic tube. The addition of a carrier such as tRNA or poly A at a concentration of 0.1 to 1.0 mg/mL can help to avoid this issue. (Please note that this concentration is just a suggestion, it is acceptable to use a higher concentration if your application requires.)
- 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.
Resuspending eBlocks Gene Fragments
Most eBlocks Gene Fragments come resuspended in 20 μL of nuclease-free water (concentration: 10 ng/μL), so there is no need to resuspend. If you elected to receive your eBlock plate dry, please follow the instructions above for resuspension.
Quantifying gBlocks Gene Fragments
Gene fragments are delivered in the following amounts:
- gBlocks Gene Fragments are delivered at amounts of 250–1000 ng
- gBlocks HiFi Gene Fragments are delivered at 1000 ng
- eBlocks Gene Fragments are delivered at 200 ng
For quantification, we recommend using methods designed for small sample volumes, such as those using the NanoDrop™ (Thermo Fisher Scientific). 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.
- Most find that when they order gene fragments resuspended by IDT, for example in plates, that quantification is not necessary.
Calculating copy number
It is often necessary to dilute the resuspended Gene Fragment to a specific copy number/µL. The molecular weight and fmol/ng conversions for each dsDNA fragment are provided on the spec sheet provided with the fragment (Figure 1).
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 IDT 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
Looking for other helpful tools? Check out IDT’s DNA Cloning guide.
IDT’s Codon Optimization Tool converts the DNA or protein sequence from one organism for expression in another. Discover the best sequence option by screening and filtering sequences to lower complexity and minimize secondary structures.
For more product information learn more about IDT’s Genes and Gene Fragments