GENECLEAN^® Protocols
BIND - WASH - ELUTE

The GENECLEAN^® Kits are used to:

• Remove and Purify DNA from Any Type or Grade of Agarose
• A Substitute for Phenol Chloroform Extraction-Alcohol Precipitation
• Remove Restriction and Modifying Enzymes (Kinases, Phosphatases, and Polymerases)
• Desalt Solutions of DNA; Ideal for Changing Buffers Without Precipitation
• Desalt Ligation Reactions Prior to Electroporation into Bacteria
• Desalt Reactions If Different Buffers are Required.
• Prepare DNA for Any Reaction.
• Clean Up Any Reaction Mixture
• Prepare Transfection-Quality DNA for In Vitro or In Vivo Work
• Eliminate Impurities such as small RNA and Proteins
• Prepare "Mini" Plasmid Preps
• Remove Ethidium Bromide (ref. 11)
• Remove Residual Phenol, Chloroform, or Ether
• Remove Unincorporated Nucleotides from Labeling Reactions
• Remove Primers from PCR Reaction Products and Excess Linkers After Ligation and Cutback
• Facilitate the Subcloning Process

All procedures require no more than 20 minutes

1. Add 3 volumes of NaI to DNA solution.
2. Add GLASSMILK Suspension.
3. Pellet GLASSMILK/DNA complex. (5 seconds)
4. Wash pellet with NEW Wash.
5. Elute DNA with water, TE, or Elution Solution.

1. Excise band from agarose gel.
2. Add 3 volumes of NaI and incubate at 55°C to melt gel.
3. Add GLASSMILK Suspension.
4. Pellet GLASSMILK/DNA complex. (5 seconds)
5. Wash pellet with NEW Wash.
6. Elute DNA with water, TE, or Elution Solution.

1. Excise band from agarose gel.
2. Add 1/2 volume of TBE Modifier and 4.5 volumes of NaI and incubate at 55° C to melt gel.
3. Add GLASSMILK Suspension.
4. Pellet GLASSMILK/DNA complex. (5 seconds)
5. Wash pellet with NEW Wash.
6. Elute DNA with water, TE, or Elution Solution.

1. Excise DNA band from ethidium bromide-stained agarose gel with a razor blade using long-wave UV light for as short a time as practical. Weigh agarose: Determine approximate volume of gel slice by weight (0.1 g equals approximately 100 µl) and then transfer to a plastic tube. Do not use glass as the DNA will bind to it. If the gel slice weighs less than 0.4 g, use a 1.5 ml microcentrifuge tube; if the gel slice weighs more than 0.4 g, use a larger tube. (The volume of tube needed in ml will be 3 to 4 times the weight of the gel slice). It is not necessary to crush the gel, but large pieces can be sliced into roughly 2mm cubes to facilitate gel dissolution during the next step.
2. Add 3 volumes of NaI solution: The volume of NaI to add to a solution of DNA (or agarose gel piece that contains DNA) should be approximately 3 times the volume of the solution (or weight of gel slice). This keeps the final concentration of NaI above 4 molar. If agarose is present, place the tube in a 45°C to 55°C water bath incubator. The plastic BioFloat contained in the GENECLEAN Kit can be used for this purpose. After a minute or two, mix the contents of the tube and return it to the water bath. After about five minutes the agarose gel should be completely dissociated. Carefully observe the solution while mixing contents to be certain all agarose has dissolved. If it has not completely dissolved, continue incubation for a minute or two and check again.
   
   When working with agarose gels in TBE buffers, add 1/2 volume of TBE Modifier and 4.5 volumes of NaI to a given volume of agarose. Incubate at 45° to 55°C to melt gel, as described above.
3. Add GLASSMILK: Resuspend GLASSMILK in GENECLEAN and GENECLEAN II Kits by vortexing for about 1 minute. For EZ-GLASSMILK, shake a few times by hand to resuspend silica particles before opening bottle.

How Much GLASSMILK® to Use:

Add quantity of GLASSMILK suspension according to the following considerations:

The amount of GLASSMILK that should be used is based on the mass of DNA that is contained in the NaI solution. 1 µg of GLASSMILK will bind 1-2 µg of DNA. Also, because a DNA molecule must collide with a silica particle to bind and collision frequency follows first order kinetics, by keeping the volume low and the particles in motion, the binding efficiency will be maximized.

Guidelines:

Add 5 µl of GLASSMILK suspension to preps containing 5 µg or less of DNA. Add an additional 1 µl for each 0.5 µg of DNA above 5 µg. If the solution volume is 0.5 ml, use a minimum of 10 µl of GLASSMILK; if 1 ml, use a minimum of 20 µl.

Binding DNA to GLASSMILK: After adding the GLASSMILK to the solution, mix and incubate at room temperature for 5 minutes to allow binding of the DNA to the silica matrix, mixing every 1-2 minutes to ensure that GLASSMILK stays suspended with the particles in motion. It is usually not necessary to continue this incubation for longer than 5 minutes. There are instances, however, when longer binding times can increase recoveries. If the volume of the binding reaction is greater than 1 ml, allow at least 15 minutes binding time while frequently mixing or place on a mechanical rocker or rotation wheel to keep the silica particles in suspension.

Scaling up the Process: The GENECLEAN Process can be scaled up, but test some of the DNA first on a small scale to determine recovery efficiency before committing a large amount to a gel. When working with preparative size gel pieces (>1 g), cut close to the band to minimize agarose. Melt the gel at room temperature by placing on a rocker or rotator for about 20 minutes after adding 3 volumes of NaI and at least 100 ul of GLASSMILK/g of agarose. Allow DNA to bind for 1-2 hours on a rotator at room temperature. Save the NaI after spinning down the GLASSMILK/DNA complex. If DNA remains in the NaI and does not bind to the matrix, it can be precipitated. Add 1/3 volume of acetone; incubate 60 minutes at room temperature and spin down the precipitated DNA. Some agarose will pellet with the DNA, but the DNA can be dissolved in water and the agarose spun out.

4. Pellet the silica matrix with the bound DNA. Spin in a microcentrifuge for approximately five seconds at full speed (but not greater than 14,000 x g). If the suspension is in a larger tube, spin it in a benchtop or other centrifuge for a minute or two to pellet. Save the supernatant aside in case all the DNA did not bind to the GLASSMILK. Resuspend the pellet in NEW Wash (~500 ul) and transfer to 1.5 ml microcentrifuge tube or spin filter ("with SPIN" protocol) for efficient handling.
   
   Note: If spin filters are not used, be sure to completely remove the NaI supernate for efficient washing. This is accomplished by first decanting the supernatant following centrifugation followed by a pulse spin and the use of a small bore pipet to remove the remaining solution.

Optional wash with NaI: The pellet can be resuspended with 200 to 400 µl of NaI stock solution to dissolve any agarose that did not dissolve or when using >2% gels. Place suspension in the 45°C or 55°C water bath for a few minutes and then pellet as in step 4. Discard NaI wash supernatant. This step is usually not necessary if care is exercised in dissolving all the agarose.

5. Wash pellet 3 times with NEW Wash: Add approximately 10 to 50 volumes (200 to 700 µl is convenient) of NEW Wash to the pellet. The consistency of the pellet is different in NEW Wash than in aqueous solutions and is somewhat resistant to resuspension. Resuspend the pellet in the wash by pipeting back and forth while digging into the pellet with the pipet tip. After it is resuspended, spin for 5 seconds in the centrifuge and discard the supernatant. Repeat the wash procedure two more times.
   
   After the supernatant from the third wash has been removed, spin the tube again for a few seconds and remove the last bit of liquid (with a small bore pipet tip) to avoid diluting the eluate with NEW Wash.
   
   Dry the pellet: Although not usually necessary, the pellet can be dried to eliminate the small amount of ethanol that is trapped in the void volume. Leave the cap open for 5-10 minutes or place tube under vacuum for 2-5 minutes.
6. Elution of DNA from GLASSMILK: Resuspend washed, white pellet with water, TE, or Elution Solution. The pellet easily resuspends in a volume that is equal to the volume of the GLASSMILK added. Centrifuge for about 30 seconds to make a solid pellet. Carefully remove the supernatant containing the eluted DNA and place in a new tube. Approximately 80% or more of the bound DNA that will elute will do so in this first step. A second elution can be done resulting in an additional 10-20% recovery of eluted DNA. A third elution normally does not add more than 1% to the total DNA yield. White pellets can be discarded after eluting DNA.

GLASSMILK® "Fines" in eluted DNA: It is often difficult to remove the last bit of eluate from the top of the pellet without carrying a small amount of the insoluble silica matrix with it. This carried-over matrix does not normally interfere with subsequent use of the DNA, and the DNA will not bind to it in less than 3M salt. To avoid transferring residual silica during subsequent use of the DNA, centrifuge the tube for a few seconds before removal of an aliquot from the upper part of the liquid. Use care to avoid adding silica particles to PCR reactions as they may interfere with the reaction. The use of the GENECLEAN "with SPIN" protocols will eliminate carry-over of silica particles in the cleaned DNA.

1. Desalting.
2. Eliminating RNA, Enzyme Activity, and Proteins.
3. Clean Up Any Reaction Mixture.
4. Elimination of Residual Organic Solvents.
5. A Rapid ssPhage DNA Isolation Protocol.
6. Elimination of Primers, Linkers, Adaptors.
7. Elimination of BAP, CIP, SAP:
8. Elimination of Unincorporated Radioactive Nucleotides from Nick Translation, End Label, Random Primed, or Fill-In Enzyme Reactions.
9. Use of GENECLEAN in "Quickcloning" Methods; Subcloning Strategies.
10. A Rapid Miniprep Protocol.
11. Double GENECLEAN for Cloning PCR Products, etc.
12. See Also Application Chart (pg. 332) & Selected Literature References (pg 341) for Additional Uses of GENECLEAN Kits

1. Desalting.

   The GENECLEAN process is ideal when the use of multiple enzymes with incompatible buffers is required (see Subcloning Stratagies 9). It is also useful for the removal of salts and other contaminants from a ligation reaction prior to transforming competent bacteria by electroporation. Trace amounts of salts will cause arcing that adversely affects the transformation efficiency. In preparing DNA for electroporation, an 80% ethanol wash is recommended after the NEW Wash and before elution in water. Furthermore, the GENECLEAN process is well suited for the removal of phosphates after hydroxylapatite (HAP) chromatography. Due to the nature of HAP, phosphate buffers are used introducing the problem of eliminating them after HAP use without losing significant amounts of DNA. Ethanol precipitation cannot be used because the phosphate will co-precipitate with the DNA. For desalting, use Protocols A or E.

2. Eliminating RNA, Enzyme Activity, and Proteins.

   The GENECLEAN process is an excellent method for removing proteins or RNA. Proteins and most larger RNA species do not bind to the silica matrix and are eliminated during the wash. Since protein and/or RNA do not compete for binding sites on the silica matrix, relatively large quantities of enzymes or other proteins are quickly and easily eliminated from, for example, a ligation reaction mix before bacterial transformation or before adding a second restriction or other enzyme to a reaction mix that may pose a competition problem or require a different buffer. Use Protocols A or E.

3. Clean Up Any Reaction Mixture.

   The GENECLEAN process is an ideal substitute for phenol/chloroform extraction-alcohol precipitation. The resulting DNA is ready for any downstream application. Use Protocols A or E.

4. Elimination of Residual Organic Solvents.

   Solutions of DNA that have been extracted with phenol and/or chloroform and/or ether contain residual organic compounds that sometimes interfere with further uses of the DNA as an enzyme substrate. This problem is not always solved by alcohol precipitation. Use of the GENECLEAN process rapidly eliminates these organic solvents without the need for alcohol precipitation and its associated losses.

5. A Rapid ssPhage DNA Isolation Protocol.

   The GENECLEAN procedure can be incorporated into small-scale, single-stranded bacteriophage DNA isolation protocols. Because phenol/chloroform is usually used to lyse the phage, the GENECLEAN process helps to rid the final DNA preparation of these solvents. The solution is less inhibitory when added to polymerase or other enzyme reaction mixtures, thus helping to maximize sequencing results.

   1. Pellet cells from 1.5 ml of a culture of cells producing M13 or other ssDNA phage.
   2. Transfer 1 ml of supernatant to new tube. Avoid transferring any host cells to prevent contamination with host cell DNA and RNA.
   4. Add PEG solution (20% PEG 8000, 2.5 M NaCl, pH3.5). Incubate at room temperature for 10 minutes and spin for 10 minutes to precipitate phage.
   5. Resuspend small phage pellet in 50 µl of TE.
   6. Lyse phage in one of two ways:
      1. Add an equal volume of buffer-saturated phenol, vortex briefly and spin for 2 minutes. Remove upper phase.
      2. Add 50 µl of formamide, mix, and heat at 55°C for 15 minutes.
      3. Proceed with GENECLEAN Protocol A to purify the ssDNA.







6. Elimination of Primers, Linkers, Adaptors.

   DNA smaller than 200 bp will typically be recovered at less than optimal efficiencies. This property of the GENECLEAN Kits is one of its most useful attributes, making it convenient for recovering large DNA species and leaving behind small DNAs, such as excess linkers after cut-back, excess primers from PCR reaction products (See Double GENECLEAN Procedure 11 on pg. 327 for efficient cloning of PCR reaction products), and unincorporated nucleotides in labeling reactions. Follow Protocols A or E to eliminate primers, linkers, or adaptors.

7. Elimination of BAP, CIP, SAP.

   After dephosphorylation reactions, heat reaction tube to 75°C for 15 minutes and follow Protocol A or E to eliminate dephosphorylation enzymes.

8. Elimination of Unincorporated Radioactive Nucleotides from Nick Translation, End Label, Random Primed, or Fill-In Enzyme Reactions.

   The GENECLEAN process is convenient for eliminating unincorporated radioactive nucleotides from labeling reactions. The Label-Block reagent included in the GENECLEAN III Kit minimizes high avidity binding of unincorporated nucleotides to GLASSMILK. The DNA is eluted into water or low salt buffer and is ready for subsequent enzyme reactions, hybridization, or other manipulations. When these procedures are done with the GENECLEAN Kits that do not contain Label-Block, the GLASSMILK pellet retains some unincorporated nucleotides labeled after elution (Label-Block minimizes this), but the labeled product elutes from the GLASSMILK. However, when removing labeled DNA from a gel where an excess of unincorporated nucleotides are not present, it is recommended that Label-Block be used to prevent excessive high avidity binding of labeled product DNA to the GLASSMILK. Use Protocol D. See pg. 311 for using GENECLEAN with non-radioactively labeled nucleotides.

9. Use of GENECLEAN® in "Quickcloning" Methods.

   Some "quickcloning" methods (ref. 8) call for the mixing of different DNA species that have been separated in low-melting point agarose gels. Cut the DNA bands from the gels. Mix. Melt the agarose. Add ligase and the agarose regels at the ligation reaction temperature. After ligation, remelt the gel; dilute and use to transform competent host cells. The "quickcloning" procedures described here can utilize any commercially available high or low-melting point agarose by incorporating the GENECLEAN process. When the DNA bands in agarose are combined, they are solubilized at a lower temperature with NaI, co-purified on GLASSMILK, and eluted with water. Because inhibitory components of agarose are eliminated during the NEW Wash steps, it is not necessary to heat the ligation reaction to 70°C to melt low-melt agarose before transformation. Concern with the expense or purity of different sources or batches of low melting-point agarose to make use of "quickcloning" techniques is eliminated. Use Protocols B or F.

   Subcloning Strategies.

   1. Single-Site Cloning
      1. Cut vector with restriction enzyme and dephosphorylate with CIP, BAP, or SAP. If restriction enzyme buffer is not compatible with that of dephosphorylase, cut with enzyme; GENECLEAN, and then dephosphorylate.
      2. Cut insert with the same restriction enzyme.
      3. Run both vector and insert on a 0.5 - 2% agarose gel and stain with ethidium bromide.
      4. Excise both vector and insert bands; combine and GENECLEAN.
      5. Elute DNA in 10 µl of water. Leave cap open for 15 minutes to evaporate residual ethanol, if any. Add 2 µl of 10x ligation buffer and 1 µl of T4 DNA ligase. Ligate for 1 - 3 hours at room temperature or overnight at 15°C and transform competent bacteria using either heat shock or electroporation.
   2. Directional Cloning:
      1. Cut vector with two different restriction enzymes. If enzyme buffers are not compatible, cut with first enzyme; GENECLEAN; and then cut with second enzyme.
      2. Cut insert with the same restriction enzymes.
      3. Run both vector and insert on a gel; excise bands, combine and GENECLEAN.
      4. Ligate, as above 1.e.
   3. Adding Linkers and Adaptors:
      1. Cut DNA with a restriction enzyme.
         • blunt end: no further treatment necessary.
         • 3' protruding end: remove using T4 DNA polymerase.
         • 5' protruding end: fill with klenow.
      2. GENECLEAN and ligate (as in 1.e.) to linkers/adaptors.
      3. GENECLEAN again (Protocol A or E) to eliminate excess unligated linkers/adaptors.
   4. Multi-Fragment Cloning:
      1. Design ligation strategy such that there is a single way the various fragments can assemble to give the desired product and cut with the appropriate restriction enzymes. GENECLEAN between reactions if enzyme buffers are not compatible.
      2. Separate fragments on a gel; excise bands; combine and GENECLEAN using Protocols B or F.
      3. Ligate as in step 1.e. Up to 5 fragments can be ligated simultaneously to give the desired clone:


w_________v + v____________x + x___________y + y__________z + z_____w


10. Rapid Miniprep Protocol Using GENECLEAN®.

    Many miniprep protocols require RNase to digest the large quantity of RNA that is co-purified with the plasmid DNA and can mask plasmid bands during screens. After RNase digestion, the enzyme is often eliminated by phenol extraction and alcohol precipitation. Using GENECLEAN in place of the RNase and clean-up steps is much more rapid and results in equivalent yields. Also, the DNA, if resistant to restriction enzyme digestion, causes a labor intensive screen to fail. This is avoided by including the 15-minute GENECLEAN process in the isolation protocol as described below.

    1. Grow 2 ml unamplified transformant cultures overnight in rich medium with appropriate selection and adequate aeration. (Circlegrow® medium available from BIO 101 is excellent for obtaining large yields of plasmid DNA from small volume cultures. See pg. 212.)
    2. Pour about 1.4 ml of each culture into a 1.5 ml microcentrifuge tube.
    3. Pellet cells in microcentrifuge for 15 to 30 seconds.
    4. Discard supernatant and resuspend cell pellet in 100ml of STET (8% sucrose, 50mM Tris pH 8, 50mM EDTA, 5% Triton X100).
    5. Add 10 µl of lysozyme (freshly made at 10 mg/ml water) to each tube and place on ice for 10 minutes.
    6. Punch a hole in each tube lid with a hot needle or uncap each tube. Place tubes in boiling water bath for no longer than 2 minutes. The tubes can be floated in the boiling water using the BioFloat supplied with the GENECLEAN Kits.
    7. Centrifuge for 15 minutes, preferably at refrigeration temperature.
    8. Remove 30-50 µl of supernatant and transfer to another tube. Avoid transferring the clear layer of viscous genomic DNA that constitutes the top of the pellet. Discard pellets.
    9. Proceed with Protocol A or E on the supernatant.

    Average yield: 2-5 µg of plasmid DNA from 1.5 ml of cultured cells, depending on growth properties of the plasmid and host cell.

    
    
    
    
    

    For large screens, see pg. 34 for the economical Miniprep Express which includes the protocol below in addition to an alkaline lysis protocol and is dedicated to minipreps for screens. The Miniprep Express contains sufficient reagents for 1250 preps. BIO 101 provides a complete line of Plasmid Prep Kits specifically designed for extracting plasmid DNA for all purposes. See pg. 33 in this catalog.

11. Double GENECLEAN for Cloning PCR Products.

    Blunt-End Cloning of PCR Products Products of thermostable DNA polymerase reactions contain "ragged ends" due to non-template nucleotide addition reactions (2) which interfere with blunt-end cloning. Efficient cloning of these products can be performed by "building in" flanking restriction sites or by "filling in" or "cutting off" the non-template additions. Ends can also be modified for "direct cloning" techniques (3,4) that do not require ligation of PCR product to vector before transformation. These procedures are often inefficient due to excess primers, enzymes, and salts present in reaction mixtures causing the frequency of desired clones in total transformants to be low or non-existent. However, by incorporating the "Double GENECLEAN" Procedure, the cloning efficiency of PCR and other amplified products is increased as much as 100 fold (4,5), resulting in a high percentage of transformants containing the correct insert.

    1. After PCR, transfer reaction mixture to new tube; leave oil behind, if used.
    2. GENECLEAN First Time: Use Protocol A or E with 15 µl of GLASSMILK and elute in 50 µl of water or Elution Solution.
    3. Modify Ragged Ends:
       To make ends flush for blunt-end cloning: Make 100 µl reaction volume using 25 µl of PCR reaction eluate from step 2, 10 µl 0.5 M Tris pH 7.5, 0.1 M MgCl₂, 10 mM DTT, 0.5 mg/ml BSA, 200 µM dNTPs, 1mM rATP, and 10 units each of E. coli DNA Polymerase I and T4 polynucleotide kinase. Incubate at 370°C for 1 hour. Stop with 1 µl 0.5 M EDTA, pH 7. (Use Pol I instead of Klenow to make use of both exo activities and include kinase if primers were not phosphorylated)
    4. GENECLEAN Second Time: Same as step 2. Use 10 µl GLASSMILK and elute in 25 µl of water or Elution Solution. No further purification is necessary. Ligate into a vector that has dephosphorylated "blunt ends" and transform competent cells.
    
    

    Cloning PCR Products with built-in restriction sites

    1. Perform PCR reaction with primers that will result in restriction sites built in. Perform steps 1 and 2 as in A.
    2. Cut with appropriate restriction enzyme(s) to create overhanging sites for cloning.
    3. GENECLEAN Second Time: Same as step A.4 above. Ligate into appropriately cut and dephosphorylated vector.

• lane 1: 7.5 µl DNA from the 10 µl aliquot in step 1 not purified with GENECLEAN. This equals the amount of DNA that was purified
• lane 2: 10 µl first elution (step 10)
• lane 3: 10 µl second elution (step 11)
• lane 4: 10 µl NEW Wash (step 9, add Ficoll®, sucrose or glycerol to keep the NEW Wash in the well)
• lane 5: GLASSMILK pellet from step 11 resuspended in running buffer
• lane 6: 10 µl of the precipitated NaI supernatant after GLASSMILK absorption (step 7)
• lane 7: 10 µl of the precipitated NaI/DNA solution before GLASSMILK is added. (This is the 10 µl aliquot removed in step 3 before adding GLASSMILK, and contains 1/3 the quantity of DNA that was exposed to GLASSMILK)
  
  
  
  
  
  

  The results of the gel should show the fate of DNA during the GENECLEAN procedure. Most of the DNA should be in the first elution (lane 2), some (approximately 10%) in the second elution (lane 3), and none in NEW Wash or NaI after adsorption (lanes 4 and 6, respectively). If DNA is seen in lane 6, it indicates that it did not all bind to the GLASSMILK. DNA in lane 4 indicates loss during the NEW Wash step. The relative quantities of DNA in each elution will indicate efficiencies during this step. If there is any DNA that would not elute from the GLASSMILK by diffusion, it may do so by electroelution (lane 5). (Note: GLASSMILK will fluoresce slightly in the well. This is not DNA). The results of this rapid kit test normally show a recovery of 70% or more in the first elution.

  Low Transformation Efficiencies: Transformation or ligation efficiencies can be enhanced by mixing the insert and vector DNA and then using Protocol A or E to purify them together. Often transformation can be increased by using Protocol A or E on the ligation reaction mixture before transformation to eliminate inhibitors of transformation that are present in some ligation reactions. If a ligase is sensitive to traces of ethanol, the GLASSMILK/DNA pellet can be dried after the last wash to eliminate ethanol before elution. UV exposure of agarose gels can cause a dramatic decrease in transformation efficiency. It is important to use long-wave UV for the shortest possible time when cutting DNA bands from gels. Often failure to get transformants or clones of interest is due to the failure of restriction enzymes to cut DNA before ligation. This is especially difficult to detect if the enzyme is used to cut off a small piece that cannot be resolved on a gel. Often the use of Protocol A or E on the DNA before cutting with a restriction enzyme(s), especially if the DNA has been phenol/chloroform extracted, will allow the enzyme to act on the substrate more efficiently; in most cases Protocol A or E can be used in place of organic extraction, thus eliminating the inhibiting effect of traces of organic solvents on subsequent enzyme activities. Because the melting temperature of dsDNA decreases with temperature in high salt concentrations (7), AT-rich fragments may denature during the gel melting step. This may reveal itself by multiple bands in a gel after removal of a single band and is caused by incomplete renaturation of denatured dsDNA. Single-stranded species can be renatured to dsDNA by heating and slow cooling. The gel melting step can be done at lower than 45° - 50°C (never at room temperature) by placing the gel slice in NaI and rotating the tube on a wheel for 15 minutes or until the agarose is "melted".

  
  
  
  References

  Many of the principles of the GENECLEAN procedures described here are based on the data of Vogelstein and Gillespie, ref. 6. See also ref. 7 for discussion of the effects of chaotropic salts and temperature on DNA stability.

  1. Wilson, V.G. (1988) A rapid procedure for removal of excess linkers. BioTechniques 6, 733.
  2. Clark, J.M. (1988) Novel non-templated nucleotide addition reactions catalyzed by prokaryotic and eukaryotic DNA polymerases. Nuc. Acids Res. 16, 9677.
  3. Hemsley, A. et al. (1989) A simple method for site-directed mutagenesis using the polymerase chain reaction. Nuc. Acids Res. 17, 6545.
  4. Aslanidis, C. and de Jong, P.J. (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nuc. Acids. Res. 18, 6069.
  5. Starr, L. and Quaranta, V. (1992) An Efficient and Reliable Method for Cloning PCR-Amplification Products: A Survey of Point Mutations in Integrin cDNA. BioTechniques 13, 612-618. (Call BIO 101 for reprint).
  6. Vogelstein, B. and Gillespie, D. (1979) Proc. Nat. Acad. Sci., USA 76, 615.
  7. Hamaguchi, K. and Geiduschek, E.P. (1962) J. Amer. Chem. Soc. 84, 1329.
  8. Struhl, K. (1985) BioTechniques 3, 452.
  9. Wilson, V.G. (1988) A rapid procedure for removal of excess linkers. BioTechniques 6, 733.
  10. Smith, L.S., Lewis, T.L. and Matsui, S.M. (1995) BioTechniques 18, 970.
  11. Glazer, A.N., Peck, N., and Mathies, R.A. (1990) Proc. Nat'l Acad. Sci., USA. 87, 3851.
  
  
  
  Selected GENECLEAN Applications and References
  
  Copyright ©1997 BIO 101, Inc. All Rights Reserved
  
  

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