MERmaid^® Kit Protocol
Revision No. 1005-999-6J03W

The MERmaid kit is designed as a Complete System to rapidly purify small quantities of low molecular weight single and double stranded DNA oligomers. The kit will remove and purify DNA between 10 bp and 200 bp from agarose or solution. When purifying DNA from agarose, especially if <50bp in length, we highly recommend that all MERmaid kit reagents be used. Substituting other agaroses or electrophoresis buffers may result in less than optimum yields. In any case, do not use TBE buffers. (For more efficient recovery of DNA species greater than 200 bp in length use one of the GENECLEAN Kits from BIO 101).

To Purify Oligomers from Solution:

1. Determine volume of DNA solution.
   1. Add 3 volumes of High Salt Binding Solution.
2. Vortex GLASSFOG vial until resuspended. Add 5-8 µl per µg of DNA in solution.
   1. Incubate at room temperature for 5-15 minutes.
   2. Efficient binding is dependent upon vigorous mixing during this incubation period. Significant increases in yield, especially with shorter length species of DNA, are often obtained by vortexing the tube during the entire binding period.
3. Centrifuge in a microcentrifuge at high speed for a few seconds to pellet the GLASSFOG.
   1. Remove supernatant and save aside.
   2. Option: Wash pellet with 200 µl of High Salt Binding Solution. Spin tube for one or two seconds and remove last bit of supernatant with small bore pipet. Note: when purifying DNA from agarose, this wash is necessary for removing traces of agarose.
4. Add 300 ml of Ethanol Wash and resuspend the GLASSFOG pellet by vortexing for a few seconds. The wash effectively removes salts or other compounds that will inhibit enzymes.
   1. Centrifuge briefly and discard supernatant.
   2. Repeat Ethanol Wash one or two more times. After last wash, spin tube for a second or two and remove remaining liquid with small bore pipet.




OPTION: Pellet can be dried under a vacuum for a few minutes to remove traces of ethanol




5. Elute oligomer DNA from GLASSFOG by resuspending pellet in a small volume of water. A convenient elution volume is equal to the volume of GLASSFOG added in step 2.
   1. Incubate at 45-55° for 5 minutes.
   2. Centrifuge for 1 minute.
   3. Transfer supernatant to new tube.
   4. Repeat elution step once and combine the two elutions.

From Gels: Running low MW DNA in Agarose
The MERmaid Kit contains a sample of BIOGEL agarose and a special gel buffer to ensure both good resolution of low MW DNA bands and good recoveries of DNA. BIOGEL is a BIO 101 product consisting of a highly pure agarose formulation designed to be less brittle than other types of agarose that will resolve low MW DNA bands. To resolve DNA species less than 50 bp in length, use 4-6% Biogel agarose. For DNA 50 to 100 bp, use 3% Biogel; for longer than 100 bp, use 2% Biogel.

To Conserve BIOGEL Agarose:
Cast a 1% agarose gel using any good quality DNA electrophoresis grade agarose. Cut out and remove a section of the gel below one lane, including well, for each sample you will be testing. Cast a BIOGEL (equal in volume to the section removed) into the resulting frame after removing the cut section. Ethidium bromide (0.5 mg/ml) can be added to the agarose before casting. Gels wrapped in saran wrap can be stored at 4°C for several days before or between uses.

When casting and running the agarose gels, use the MERmaid Electrophoresis Buffer supplied in the kit for best recovery of DNA from the gel, especially if 50bp or less in length. Dilute the 50x concentrated solution to 1x with water before use. Enough 50x concentrate is included in the kit to make 5 liters (200 ml with sample kit size) of 1x Electrophoresis Buffer. If other electrophoresis buffers are used, soak the gel slice in a solution of 1 x MERmaid Electrophoresis Buffer for 15 minutes before removing the DNA from it with the MERmaid Kit. Avoid TBE buffers, if possible, because recovery of DNA from agarose is adversely affected by the presence of borate. Gels run in the presence of 1/2 to 1 mg/ml ethidium bromide will allow visualization of single-stranded DNA.

Removal of low MW or oligomer DNA from Agarose:

1. Use BIOGEL and MERmaid Electrophoresis Buffer for best results. For sharper resolution bands, limit electrophoresis to 10-15 minutes at high voltage to minimize diffusion of DNA in agarose. Cut the desired DNA band from agarose gel and place in a microcentrifuge tube. Add 3 volumes of High Salt Binding Solution.
2. Add 8 µl of resuspended GLASSFOG per µg of DNA to tube. (Binding capacity is approximately 1 µg DNA per µl of GLASSFOG¨, but it is best to add excess to increase binding kinetics.) Vortex tube continuously for 10 minutes. The gel will "melt" rapidly at room temperature and the DNA will bind to the GLASSFOG most efficiently under vigorous mixing conditions. Continue, starting with step three (3) in the protocol for removing oligomers from solution and include the optional salt wash.

To remove oligomer DNA from Polyacrylamide Gels:

Method A
Cut out the band and cut or crush the gel into small pieces. Soak the gel pieces in approximately twice the volume of High Salt Binding Solution for 20 minutes at 60°C. Centrifuge and remove the supernatant, avoiding gel pieces. Repeat soak with one volume of High Salt Solution and pool supernatants. Add GLASSFOG and continue with the MERmaid Kit protocol for solutions.

Method B
Cut out the band from the polyacrylamide gel and place in a slit in a Biogel agarose gel. Electroelute the DNA from the polyacrylamide into the Biogel agarose. Excise the band from the Biogel and purify the DNA using the appropriate Mermaid Kit procedure.

1. Add 3 volumes of High Salt Binding Solution to DNA.
   1. Add 3µl High Salt Binding Solution per µl of DNA solution.
   2. If working with agarose, add 3µl of High Salt Binding Solution per mg of gel slice.
2. Add GLASSFOG; vortex; transfer suspension to a spin filter; spin.
   1. Add 5-8µl of GLASSFOG suspension per µg of DNA.
   2. Vortex tube continuously for 10 min to melt gel and allow for DNA binding. (Do not vortex in SPIN Filter because the filter will tear easily)
   3. Transfer suspension to a SPIN Filter. The SPIN Filter capacity is 750µl, multiple spins are necessary when working with larger volumes of suspension.
   4. Spin in a microcentrifuge at full speed for 15 seconds, longer if necessary, to transfer liquid to catch tube. (For this and all wash steps, empty catch tube as needed.)




Note: Wash pellet with 200 ml of High Salt Binding Solution when removing DNA from agarose




3. Wash twice with Ethanol Wash.
1. Add 300µl of Ethanol Wash solution to the SPIN Filter.
2. Spin for 30 seconds or until SPIN Filter is emptied of wash. Repeat wash.
3. Empty catch tube and spin for 1 minute to "dry" pellet of ethanol that might interfere with subsequent reactions.
4. Transfer SPIN Filter to a catch tube and elute DNA with water or TE.
   1. Add an amount that is 1-2 times the volume of the GLASSFOG suspension used in 2a and gently resuspend the white pellet by hand, pipet, or vortex. (No more than 1-2 seconds at 1/2 speed).
   2. Spin tube for 30 seconds to transfer eluted DNA to catch tube.
   
   
   

   A second elution can increase yield by 10-20%

   
   
   
   3. Discard SPIN Filter and cap the tube. DNA in solution is ready to use without further manipulation.