FAQ: GENECLEAN

What are the differences between the GENECLEAN, GENECLEAN II, and GENECLEAN III Kits?

What is the difference between the regular GENECLEAN Kits and the GENECLEAN SPIN Kit?

What is the difference between the GENECLEAN SPIN Kits and the GENECLEAN Turbo Kits?

I got low/no recovery with my GENECLEAN Kit. What might have gone wrong?

How can I determine my GENECLEAN yield?

Do I need to do anything different when using GENECLEAN with high-molecular-weight DNA (›10 kb)?

Does GENECLEAN work on all conformations of plasmid DNA?

Can I substitute GENECLEAN NEW Wash for my GENECLEAN SPIN NEW Wash?

I ran out of NEW Wash in the middle of a prep. What can I do?

What happens if I don't remove all of the NEW Wash from the GLASSMILK pellet before eluting?

If I'm using GENECLEAN SPIN, what do I do if I have more than 5 µg of DNA or a large gel slice?

If I'm using GENECLEAN, what do I do if I have more than 5 µg of DNA or a large gel slice?

Q. What are the differences between the GENECLEAN, GENECLEAN II, and GENECLEAN III Kits?

A. The GENECLEAN, GENECLEAN II and GENECLEAN III Kits are similar in that they all use NaI, GLASSMILK, and NEW Wash to purify DNA.

• The GENECLEAN Kit (200 preps) is used for purifying DNA from solution or from TAE-buffered agarose gels.
• The GENECLEAN II Kit (300 preps) comes with an additional reagent, TBE Modifier, that enables isolation of DNA from TBE-buffered gels.
• The GENECLEAN III Kit (600 preps) has 3 further modifications or additions: 1) EZ-GLASSMILK resuspends more easily than GLASSMILK, which it replaces, but is otherwise identical; 2) Label Block prevents irreversible binding of 32P-labeled DNA to GLASSMILK when purifying from an agarose gel; 3) Elution Solution is simply nuclease- and pyrogen-free water.

Q. What is the difference between the regular GENECLEAN Kits and the GENECLEAN SPIN Kit?

A. The GENECLEAN Spin Kits have the same basic steps as the GENECLEAN Kits: Bind, Wash, and Elute. The main differences between the kits are the following:

• GENECLEAN Spin uses guanidine thiocyanate for the chaotropic salt instead of the NaI found in GENECLEAN.
• Instead of having the chaotropic salt and the GLASSMILK as separate components, they are combined into one suspension (GENECLEAN SPIN GLASSMILK).
• There is a built-in TBE Modifier, so the kit can be used with both TAE- and TBE-buffered gels.
• All 3 steps are done in a SPIN Module, allowing for rapid binding, washing, and elution with minimal manipulation of the GLASSMILK/DNA pellet. The amount of agarose is limited to 300 mg (300 µl liquid volume) to avoid overflowing the SPIN Module.
• Because a fixed volume of GENECLEAN SPIN GLASSMILK is added, the binding capacity of the kit is 5 µg/prep (5 µg DNA/ 400 µl GENECLEAN SPIN GLASSMILK).
• While TE or autoclaved water can still be used to elute the DNA from the GLASSMILK, we now supply Elution Solution, which is nuclease/pyrogen-free water.

As with the GENECLEAN Kits, low-melt agarose is not required; the kit will work with any molecular biology-grade agarose. The DNA purified using this kit is suitable for almost any downstream procedure (i.e. ligation, restriction digestion, transfection/transformation, manual and autofluorescent sequencing, etc.)

Q. What is the difference between the GENECLEAN SPIN Kits and the GENECLEAN Turbo Kits?

A. The new GENECLEAN Turbo and GENECLEAN Turbo for PCR Kits still operate under the same principals as the GENECLEAN Kits: Bind, Wash, and Elute. The main difference is that these new Turbo kits utilize a specially designed filter that has the GLASSMILK immobilized on the membranes. The irregular shape of GLASSMILK plus the thickness of the filter allow for a greater binding surface area and higher capacity than most filter-based purification systems. Each column can bind up to 10 µg of DNA. The hole in the top of the tube allows for rapid delivery of solutions to the filter and the hole in front allows for easy removal of liquid from the catch tube using a vacuum attached to a syringe.

Q. I got low/no recovery with my GENECLEAN Kit. What might have gone wrong?

A. In the GENECLEAN procedure, there are 3 basic steps:

• Binding: NaI melts the agarose and allows the DNA to bind to the GLASSMILK.
• Wash: NEW Wash cleans the DNA
• Elute: DNA is eluted from the GLASSMILK with water or TE.

1. Binding: Binding of the DNA is dependent on both salt concentration and pH. If less than 3 volumes of NaI were used, the salt concentration might not be high enough to allow the DNA to bind to the GLASSMILK. As NaI gets older or is used frequently, it can change color and the pH may change. Once the pH rises above 7.5, the binding efficiency decreases. Radioactive isotope-labeled DNA may bind irreversibly to the silica. A Label Block solution comes with the GENECLEAN III kit that is effective in preventing this. Label Block is also available separately for use with any GENECLEAN Kit. The GENECLEAN Kits are designed specifically for purification of DNA of length ›200 bp to allow for purification of larger-size DNA from smaller linkers, adapters, and primers, without the need to gel purify first. BIO 101 offers the MERmaid Kit for purification of DNA of length ‹200 bp.
2. Washing: If the NEW Wash ethanol concentration drops significantly, the DNA may elute in the wash. DNA may also elute in the wash if ethanol was not added to to the New Wash Concentrate prior to use.
3. Elution: DNA will elute from the GLASSMILK in water, TE, or other low-salt, neutral solution. If there is residual NEW Wash present, the yield may be low and the residual ethanol may interfere with many downstream applications. Be sure to remove all traces of NEW Wash. As an optional step, dry the pellet for 5 minutes prior to elution.

The troubleshooting guide in the GENECLEAN protocol can be used to determine the problem step. If you save the supernatant at each step as well as the GLASSMILK pellet, you should be able to recover the DNA from any point in the procedure.

Q. How can I determine my GENECLEAN yield?

A. DNA yield can be quantified with a fluorimeter or estimated by running the sample against a known amount of DNA on an agarose gel. Using a spectrophotometer to quantify DNA yield is not recomended for the following reasons:

1. Residual silica particles (which do not interfere with downstream reactions or uses of the DNA) can scatter UV light, affecting OD260 readings and OD260/OD280 ratios.
2. After diluting part of your sample up to the minimum volume of the cuvette, the DNA will often be too dilute to give a significant reading. For example, if you eluted 0.5 µg in 20 µl of water and diluted 2 µl of this to 200 µl, the final concentration of DNA in the cuvette would be 0.5 µg/0.02 ml x 0.01 = 0.25 µg/ml. This would give an absorbance of only 0.005, which is too low to be significant on most instruments.

Q. Do I need to do anything different when using GENECLEAN with high-molecular-weight DNA (›10 kb)?

A. Shearing can be a problem for high-molecular-weight DNA, but may be avoided by doing the following:

1. Use a wide-bore pipet and minimize agitation of the GLASSMILK/DNA pellet. For the wash steps, allow the GLASSMILK pellet to soak in the NEW Wash instead of resuspending it. When eluting, gently resuspend the pellet using a wide-bore pipet.
2. Use SPIN Modules (2080-X00) with the GENECLEAN Kits. This allows the customer to avoid shearing the DNA while it is bound to the GLASSMILK, to efficiently remove the NEW Wash, and to elute the DNA without carryover of silica.

Q. Does GENECLEAN work on all conformations of plasmid DNA?

A. YES. If a customer's yields are not satisfactory, increasing the binding time and the volume of GLASSMILK will generally increase recoveries.

Q. Can I substitute GENECLEAN NEW Wash for my GENECLEAN SPIN NEW Wash?

A. No. The two wash solutions have different salt concentrations and are made up from different concentrates. NEW Wash is prepared by diluting in water before adding 100% ethanol (as described on the bottle label), while GENECLEAN SPIN New Wash is prepared by directly adding an equal volume of 100% ethanol.

Q. I ran out of NEW Wash in the middle of a prep. What can I do?

A. For competitive reasons, we can't give out the exact composition of New Wash. However, you can make up either one of two solutions that will work nearly as well: 1) make an 80% ethanol solution using TE (10 mM Tris and 1 mM EDTA (pH 7.5); 2) make a 50% ethanol solution using TE (pH 7.5) and 100 mM NaCl.

Q. What happens if I don't remove all of the NEW Wash from the GLASSMILK pellet before eluting?

A. Most of the problems experienced by users (low yield, poor ligation, samples "floating" away when loading a gel) can be traced to residual ethanol from the NEW WASH. Remember to pulse spin, pipet off the last traces of NEW Wash. As an option, you can dry the pellet for ~5 minutes before elution.

Q. If I'm using GENECLEAN SPIN, what do I do if I have more than 5 µg of DNA or a large gel slice?

A. Because, the GENECLEAN SPIN Filter accommodates up to 400 µl of GENECLEAN SPIN GLASSMILK, a normal prep with a single loading of GENECLEAN SPIN GLASSMILK is limited to ‹5 µg DNA. To process larger amounts of DNA, the volume of GENECLEAN SPIN GLASSMILK should be scaled up proportionally (e.g. 600 µl for 7.5 µg DNA in a 600 mg gel slice) and the agarose melted in a separate tube. Transfer 700 µl to a SPIN Filter, spin for 30 seconds and repeat, making sure that the GLASSMILK/DNA complex is all pelleted on the same side of the filter. Then proceed with the written protocol.

Q. If I'm using GENECLEAN, what do I do if I have more than 5 µg of DNA or a large gel slice?

A. If working with more than 5 µg of DNA, add 2 µl of GLASSMILK for every 1 µg of DNA and proceed with the written protocol. A large gel slice can always be divided into pieces ‹300 mg that can be processed in parallel, according to the GENECLEAN protocols. Alternatively, there is a special GENECLEAN protocol for processing large gel slices (› 1 g) or large amounts of DNA (›20 µg) available from BIO 101 Technical Services.