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Troubleshooting Guide for DNA Digestion

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1. 0 1 ug l 1 3 Unsuitable DNA template or contaminated DNA solution If the enzyme is active in the control digest assay the substrate DNA solution for inhibitory contaminants in a mixing experiment with control template e g Lambda DNA danr den SD0021 Perform a control digest with two templates control template and sample template in one reaction mixture Do not exceed the optimal DNA concentration in the reaction mixture 0 02 0 1 g l e The sample template is contaminated if neither the control DNA template nor sample template is digested see 1 3 1 e The sample template is not contaminated if the control DNA template is digested but the sample template is not Poor digestion of the experimental template is caused by errors in the DNA sequence see 1 3 2 methy lation effects see 1 3 3 or structure of the DNA substrate see 1 3 4 Note Always ensure that the control DNA contains a recognition site for the enzyme present in the reaction For example there is no Not recognition site in lambda DNA continued on next page Bulk quantities and custom formulations available upon request 205 N LLI gt N LLI cc N LLI lt zz gt o Bas N lt b a od N H 206 amp Fermentas 4 f LIFE SCIENCES 1 Incomplete digestion or no digestion Table 1 26 Troubleshooting Guide for DNA Dige
2. amp Fermentas LIFE SCIENCES Troubleshooting Guide for DNA Digestion Digestion problem 1 2 3 N Incomplete Unexpected Diffuse DNA digestion no cleavage bands on gel gt digestion pattern N tu Assess en 2 1 3 1 zyme activity Star Gel shift with control activity DNA oc n 11 22 3 2 ce Inactive Active Contamination Contaminated enzyme enzyme with another reagents RE l 1 2 1 3 1 4 2 3 Suboptimal Unsuitable or Water Contamination 2 reaction contaminated contains with another T conditions DNA impurities DNA o a 1 2 1 Evaluate 2 4 N Suboptimal inhibition by Incomplete digestion template DNA DNA digestion A protocol solution it N n a 1 2 2 2 5 T Improper Inhibition No inhibition Gel shift enzyme dilution 1 2 3 1 3 1 1 3 2 2 6 Improper Contaminants Absence of Unexpected reaction in DNA recognition sites in assembly solution sites template DNA 204 1 2 4 Excess Cleavage glycerol blocked by methylation 1 2 5 Suboptimal Structure of DNA DNA concentration substrate www fermentas com 1 3 4 1 Supercoiled plasmid DNA 1 3 4 2 Proximity ends www fermentas com doubledigest site to DNA 1 3 3 1 3 4 1 3 4 3 of www fermentas com research At least two sites required 1 3 4 4 Site preference www fermentas com reviewer ISO ISO 9001 14001 Problem 1 Inc
3. ul of FastDigest restric tion enzyme per 1 ug DNA e Use the recommended reaction buffer Ensure that the glycerol concentration in the reaction mixture does not exceed 5 Reduce the incubation time For FastDigest enzymes refer to Table 1 3 on p 44 for maximum incubation times Ensure the volume of the reaction mixture was not reduced due to evaporation during incubation the resulting increase in glycerol concentration may cause star activity 2 2 Contamination with another restriction enzyme The restriction enzyme or buffer may be contaminated with another restriction enzyme due to improper han dling Use a new tube of enzyme and or buffer 2 3 Contamination with another substrate DNA The sample DNA contains a mixture of two or more different DNAs Prepare new sample of DNA e For plasmid DNA preparation pick one isolated colony of recombinant E coli and purify with GeneJET Plasmid Miniprep Kit K0503 e For PCR products check the product purity on an agarose gel If necessary purify the PCR product prior to digestion with the DNA Gel Extraction Kit K0513 2 4 Incomplete DNA digestion see 1 Different DNA structures like nicked supercoiled dimeric molecules will always show different mobility on gels compared to same size DNA size standards as an example see the picture below for migration of plasmid DNA forms 2 Unexpected cleavage pattern _ GeneRuler 1 kb DNA Ladder SM0311 Undigested
4. lic conventional restriction enzymes first digest with the mesophilc enzyme 1 h then increase the temperature and incubate for an additional hour e Ensure the volume of the reaction mixture was not reduced due to evaporation during incubation the increase in salt concentration may reduce enzyme activity For thermophilic enzymes use a heat block with a hot bonnet e g a PCR cycler 1 2 2 Improper enzyme dilution e Dilute restriction enzymes with Dilution Buffer for Restriction Enzymes B19 Restriction enzymes diluted with this buffer are stable for at least 3 4 weeks at 20 C for more information see p 170 e Never dilute enzymes in water or 10X reaction buffer e Never dilute enzymes in 1X reaction buffer in the absence of DNA 1 2 3 Improper reaction assembly e The restriction enzyme should always be the last component added to the reaction mixture e The restriction enzyme may be inactivated if added directly to a 10X reaction buffer 1 2 4 Excess glycerol in the reaction mixture e The glycerol concentration in the reaction mixture should not exceed 5 Thus the volume of the restriction enzyme added to the mixture should not exceed 1 10 of the total reaction volume e Enzymes sensitive to high glycerol concentration include Alw21I Bpil Bsp68l BspTl Eco32 Eco911 EcoRI Hin6l Hinfl Mph11031 Mva12691 and Ncol 1 2 5 Suboptimal DNA concentration The optimal range of DNA concentration in the reaction mixture is 0 02
5. nhibited by methylation of the recognition site Identify which type of DNA methylation can occur on the recognition site and determine if the methylation impairs or blocks DNA digestion with the enzyme See Digestion of Methylated DNA on p 171 and use the Tables 1 14 1 21 on pp 172 177 lf methylation impairs or blocks DNA cleavage propagate your plasmid in an E coli dam denr strain the E coli GM2163 danr den strain M0099 is available upon request with the purchase of any Fermentas product use the REsearch engine at www fermentas com research or check the Fermentas catalog for the availability of a restriction enzyme isoschizomer not sensitive to DNA methylation A restriction enzyme which requires a methylated recognition sequence Dpnl was used to digest unmethy lated DNA In the case of Dpnl the neoschizomers Bsp143 or Mbol can be used to digest non methylated Dpnl recogni tion sites Alternatively propagate your plasmid in E coli dam strains most conventional laboratory strains are dam Please refer to p 450 for an overview about the genotypes of some common E coli strains Note When PCR is carried out with standard dNTPs and non methylated primers the resulting DNA product is NOT methylated 1 3 4 Structure of substrate DNA 1 3 4 1 Supercoiled plasmid DNA Use FastDigest enzymes which are qualified for supercoiled DNA and provide specific recommendations for each enzyme see Table 1 3 on p 44 For
6. of both enzyme amp buffer diffused bands in controls 1 2 and 4 Follow the recommendations given above e Contaminated water diffused bands in controls 1 2 and 3 Bacterial or DNase contamination in improperly handled water will cause DNA degradation Use commercially available nuclease free molecular biology grade water e g R0581 N LLI gt N LLI cc N LLI lt zz gt o Bas N lt b a cist N H 208 www fermentas com www fermentas com doubledigest www fermentas com research www fermentas com reviewer
7. omplete digestion or no digestion 1 _ FastDigest amp CONVENTIONAL RESTRICTION ENZYMES Troubleshooting Guide for DNA Digestion Table 1 26 Troubleshooting Guide for DNA Digestion Possible cause and recommended solution Assess enzyme activity The restriction enzyme may lose activity due to improper storage or handling Perform a digestion reaction with 1 pg of a standard control DNA e g Lambda DNA danr denr SD0021 1 1 Inactive enzyme gt If the enzyme does not cut the control DNA e Check the expiration date e Verify that the enzyme has been stored at 20 C e Check the temperature of your freezer Do not allow the temperature go below 20 C as the enzyme may freeze and multiple freeze thaw cycles more than 3 cycles may result in reduced enzyme activity 1 2 Suboptimal reaction conditions 1 2 1 Suboptimal digestion protocol Follow digestion protocol specified for the restriction enzyme and type of substrate DNA e Use the recommended reaction buffer supplied with the restriction enzyme For double digestions with con ventional restriction enzymes follow the recommendations of the DoubleDigest engine at www fermentas com doubledigest e Use additives where required e Perform the reaction at the optimal temperature specified for the restriction enzyme refer to Table 1 8 on p 162 for data on the activity of thermophilic enzymes at 37 C For double digestions with mesophilic and thermophi
8. plasmid pUC19 2 7 kb DNA forms upper band 5 kb dimeric plasmid below less visible 4 kb nicked plasmid lowest band 1 9 kb supercoiled plasmid 3 Linearized plasmid pUC19 2 7 kb migrates according to its size I IN 2 5 Gel shift see 3 1 2 6 Unexpected recognition sites in template DNA Newly generated target sites in constructed DNA may be overlooked Recheck your DNA sequence and clon ing strategy Refer to Tables 1 23 1 24 or 1 25 for Newly Generated Recognition Sequences pp 186 201 to identify all the cleavage sites present in the substrate DNA continued on next page Bulk quantities and custom formulations available upon request 207 amp Fermentas 4 P LIFE SCIENCES Table 1 26 Troubleshooting Guide for DNA Digestion Problem Possible cause and recommended solution 3 Diffused DNA bands 3 1 Gel shift Enzyme that remains bound to the substrate DNA will affect the electrophoretic mobility of the digestion pro ducts Restriction enzymes Aarl Alol Bdal BseXI Bvel Csel Eco571 Eco57MI EcoRII Faql Gsul Lwel Mboll FastDigest Mboll Mnil FastDigest Mnil Schl Tsol Tstl are particularly prone to remaining bound to the substrate DNA This will result in a band or smear above the expected band see picture below Use 6X DNA Loading Dye amp SDS Solution R1151 for sample preparation or heat the digested DNA in the presence of 1X SDS prio
9. r to electrophoresis M GeneRuler DNA Ladder Mix SM0331 1 0 5 ug A DNA prepared for loading with 6X DNA Loading Dye R0611 2 0 5 ug A DNA prepared for loading with 6X DNA Loading Dye amp SDS Solution R1151 3 0 5 ug A DNA digested with Tsol ER1991 probe prepared for loading with 6X DNA Loading Dye R0611 4 0 5 ug A DNA digested with Tsol probe prepared for loading with 6X DNA Loading Dye amp SDS Solution R1151 3 2 Contaminated reagents Any restriction digestion reaction components may become contaminated with nucleases due to improper handling or storage Nuclease contamination causes DNA degradation which appears as diffused DNA bands ona gel Perform four control reactions without restriction enzyme ll with a new vial of buffer lll without restriction enzyme with a new vial of buffer IV with commercially available water e g Water nuclease free R0581 e Contaminated sample DNA diffused bands in all controls Re purify the DNA sample by spin column or phe nol chloroform extraction and ethanol precipitation see p 356 e Contaminated enzyme diffused bands in controls 2 and 4 The enzyme may become contaminated due to improper handling Use a new vial of enzyme e Contaminated buffer diffused bands in controls 1 and 4 Bacterial contamination of the reaction buffer will cause DNA degradation Use a new vial of buffer Store all buffers at 20 C e Contamination
10. some conventional restriction enzymes additional units are required to digest supercoiled plasmids com pletely e g 5 10 u 1 uI of restriction enzyme per 1 g of DNA check the notes in the catalog description of he enzyme or refer to the Certificate of Analysis 1 3 4 2 Proximity of the recognition sequence to the DNA ends Some restriction enzymes cleave DNA poorly if the recognition site is too close to the end of the DNA mole cule For FastDigest enzymes refer to Table 1 3 on p 44 or the product description to determine the effectiveness of restriction enzymes at the ends of DNA For conventional restriction enzymes refer to Tables 1 9 p 162 and Table 1 10 p 164 Consider direct cloning of your PCR product into a cloning vector e g CloneJET PCR Cloning Kit K1221 or InsTAclone PCR Cloning Kit K1213 Available in certain countries only 1 3 4 3 Restriction Enzyme requires at least two sites per DNA molecule to obtain optimal activity Some restriction enzymes such as Aarl Bvel Cfr42 Eam1104 Eco571 EcoRI Lwel Sfil require at least two target sites per DNA molecule for efficient cleavage for more details see Site Preferences by Restriction En zymes on p 202 If there is only one recognition site per DNA molecule add a DNA oligonucleotide containing the recognition site 1 3 4 4 Site Preferences by Restriction Enzymes The DNA sequence surrounding the recognition site may influence the efficiency of dige
11. stion Problem Possible cause and recommended solution 1 3 1 Contaminants in the DNA solution Template DNA may contain residual SDS EDTA proteins salts or nucleases Repurify the template using a spin column purification kit or by phenol chloroform extraction and ethanol precipitation see p 356 DNA A gq ratio should be 1 8 2 0 To remove EDTA and salts wash the pellet with 70 cold ethanol For reliable and reproducible plasmid miniprep purity use the GeneJET Plasmid Miniprep Kit K0503 For digestion of unpurified PCR products dilute DNA at least 3 fold in the recommended 1X restriction enzyme buffer see protocols in p 165 or p 43 for FastDigest enzymes If the template DNA has been purified using silica or resin suspensions remove all remaining particles by centrifugation for 10 min at 10 000 rpm and ensure that no resin is carried over while transferring the DNA solution into a new tube 1 3 2 The substrate DNA does not contain a recognition sequence for the restriction enzyme Re check the DNA sequence and cloning strategy Determine if the restriction enzyme selected requires more than one site per target DNA for 100 activity see also 1 3 4 3 Check literature for known site preferences for the restriction enzyme see also 1 3 4 4 If the recognition sequence had been introduced by PCR primers verify that the primer sequence contains the recognition site 1 3 3 Methylation effects Restriction enzyme is i
12. stion Some DNA sites are cleaved slowly or not cleaved at all for more details see p 202 due to the surrounding sequence Use ad ditional units 5 10 u of the restriction enzyme per 1 ug of DNA or determine if an isoschizomer has superior cleavage efficiency see Table 1 27 on p 209 or REsearch engine at www fermentas com research continued on next page www fermentas com www fermentas com doubledigest www fermentas com research www fermentas com reviewer ISOISO 1 _ FastDigest amp CONVENTIONAL RESTRICTION ENZYMES 9001 14001 Troubleshooting Guide for DNA Digestion Table 1 26 Troubleshooting Guide for DNA Digestion Problem Possible cause and recommended solution 1 4 Water contains impurities Compare you results using commercially available nuclease free molecular biology grade water e g Water nuclease free R0581 Check the quality of the water used in you lab e Check the pH and conductivity of water The pH of high quality water should be 5 5 6 0 with a resistance of gt 18 MQ e Centrifuge 10 min 10 000 rpm 1 ml of water and check if there is a visible pellet e Determine if the water contains nucleases or bacterial contamination see 3 2 for control reactions 1 Incomplete digestion or no digestion 2 1 Star activity relaxed specificity of restriction enzyme see p 206 for more details e Reduce the units of restriction enzyme not more than 10 u of restriction enzyme or 1

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