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Fig. 4.21. Temporal temperature gradient gel. Amplified mutant and wild-type alleles of exon 7 from
the cystic fibrosis gene.

Separation by TTGE run at 130 V for 5 hours in 1.25x TAE buffer on a 6 M urea/6%

acrylamide gel (37.5:1) using a temperature range of 50–60 °C and a ramp rate of 2 °C/hr. Lane 1, mutant
allele (1154 insTC); lane 2, mutant allele (G330X); lane 3, mutant allele (deltaF311); lane 4, mutant allele
(R334W); and lane 5, wild-type allele. (Samples courtesy of L. Silverman, Division of Molecular Pathology,
University of North Carolina School of Medicine)

Calculating the Run Parameters

To determine the temperature range to use with TTGE, a melting profile of the DNA

sequence should be generated using a DNA melting software program, such as Bio-Rad’s
MacMelt software. As in DGGE, the addition of a 30–40 base pair GC clamp should be added
to one of the PCR primers to insure that the region screened is in the lowest melting domain. The
temperature range for the gradient can be calculated from the melting profile graph by first
determining the lowest and highest non-GC clamp melting temperature of the DNA sequence
(See example in Figure 4.22). From the calculated low and high temperatures, the
theoretical melting temperatures can be lowered by adding urea to the gel. A denaturing urea gel
will lower the theoretical melting temperature of DNA by 2 °C for every mole of urea.

32, 33

In Figure 4.22, the theoretical melting temperature range on the DNA sequence of interest is
approximately 68 to 82 °C. Therefore, the temperature range should be 54–68 °C when using
a 7 M urea gel. TTGE gels typically use 6 M of urea, but for sequences that generate melt
profiles that require buffer temperature greater than 70 °C, higher concentrations of urea should
be used. Adding 1–2 °C to the final temperature may help to improve the resolution of some
mutations. The typical temperature range for TTGE gels are between 40 and 70 °C.

Temperature ramp rates of 1–3 °C/hr generally give the best resolution between mutant and

wild-type samples. Slower ramp rates are best, but to reduce run times for routine screening,
ramp rates can be increased empirically. The temperature ramp rate can be determined if the
desired run time or temperature range is known. The ramp rate is calculated by subtracting the
final temperature from the initial temperature and dividing by the desired run time. In Figure
4.22, if the run time is 4 hours, the ramp rate will be 3 °C/hr ([68°–54°] ÷ 4 hr = 3.5 °C/hr). A
desired run time is calculated by subtracting the final temperature from the initial temperature
and dividing by the desired ramp rate. In the Figure 4.22 example, if the ramp rate is 2 °C/hr,
then the run time will be 7 hours ([68°– 54°] ÷ 2 °C/hr = 7 hr).

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Содержание DCODE

Страница 1: ...THE DCODE UNIVERSAL MUTATION DETECTION SYSTEM Catalog Numbers 170 9080 through 170 9104 For Technical Service Call Your Local Bio Rad Office or in the U S Call 1 800 4BIORAD 1 800 424 6723...

Страница 2: ...use when operated in accordance with the instruction manual This instrument should not be modified or altered in any way Alteration will Void the manufacturer s warranty Void the EN61010 1 safety cer...

Страница 3: ...allel Gradient Gel Sandwich 23 Casting Parallel Gradient Gels 25 4 2 Introduction to Constant Denaturing Gel Electrophoresis CDGE 27 Reagent Preparation 28 Gel Volumes 30 Sample Preparation 30 Tempera...

Страница 4: ...paration 57 7 3 Gel Volumes 59 7 4 Sample Preparation 59 7 5 Temperature Controller 59 7 6 Adding the Running Buffer 60 7 7 Assembling the PTT Gel Sandwich 60 7 8 Casting PTT Gels 62 Section 8 Electro...

Страница 5: ...interlock DC current to the cell is broken when the lid is removed Do not attempt to circumvent this safety interlock Always disconnect the AC cord from the unit and the cord from the DC power supply...

Страница 6: ...eloped to analyze the presence of mutations in a DNA target The most common methods include Single Strand Conformational Polymorphism1 SSCP Denaturing Gradient Gel Electrophoresis2 DGGE carbodiimide3...

Страница 7: ...2 well 1 mm 10 cm system 2 16 well comb 1 mm 1 Comb gasket for 0 75 1 mm spacers 1 Comb gasket holder 1 Model 475 gradient former 1 Syringes 10 ml 30 ml 2 each Tubing 3 feet Luer couplings 4 Luer syr...

Страница 8: ...Electrophoresis temperature control module 1 Electrophoresis cooling tank 1 Casting stand with sponges 1 Sandwich core 1 DCode lid stand 1 Sandwich clamps 2 sets Glass plates 20 cm 2 sets Spacers 0 7...

Страница 9: ...ption Electrophoresis Tank The electrophoresis tank is a reservoir for the running buffer Electrophoresis Cooling Tank The electrophoresis cooling tank has two ceramic cooling SSCP only fingers inside...

Страница 10: ...bar fits inside the support hole of the tank The clear loading lid is a removable part that containsfourbananajackswhichfunctionasasafetyinterlock Itshouldbeleftinplaceatalltimesexceptwhileloadingsamp...

Страница 11: ...t card simplifies sandwich assembly by keeping the spacers in the correct position Comb Gasket Holder The comb gasket holder holds the comb gasket that prevents DGGE only leakage of acrylamide during...

Страница 12: ...ws the air to escape through the comb gasket holder vent port The 16 x 16 cm gel format consist of two different spacers one with the groove and injection port hole for casting and one with a short gr...

Страница 13: ...omponents when the lid is not on the electrophoresis tank Fig 3 10 Model 475 Gradient Delivery System 9 Gasket holder Comb gasket Air vent Pressure clamp Pressure clamp screw Large glass plate Small g...

Страница 14: ...is held in the holder by tightening the holder screw against the sleeve Volume Adjustment Screw The volume adjustment screw is on both sides of the syringe holder Figure 3 10 It adjusts the holder to...

Страница 15: ...perpendicular denaturing gradient gel At a low concentration of denaturant the DNA fragment remains double stranded but as the concentration of denaturant increases the DNA fragment begins to melt The...

Страница 16: ...e PCR primers called ChemiClamp primers 10 Because ChemiClamps covalently link the two DNA strands at one end they should not be used when isolating a DNA fragment which is going to be sequenced from...

Страница 17: ...ent analyzed Both 0 and 100 denaturant should be made as stock solutions A 100 denat urant is a mixture of 7 M urea and 40 deionized formamide Reagents for casting and run ning a DGGE gel are included...

Страница 18: ...p 8 200 400 bp 10 100 300 bp 0 Denaturing Solution 6 Gel 8 Gel 10 Gel 12 Gel 40 Acrylamide Bis 15 ml 20 ml 25 ml 30 ml 50x TAE buffer 2 ml 2 ml 2 ml 2 ml dH2 O 83 ml 78 ml 73 ml 68 ml Total volume 100...

Страница 19: ...l 0 05 2 Xylene cyanol 0 25 ml 0 05 100 Glycerol 7 0 ml 70 dH2 O 2 5 ml Total volume 10 0 ml Store at room temperature 1x TAE Running Buffer Reagent Amount 50x TAE buffer 140 ml dH2 O 6 860 ml Total v...

Страница 20: ...denaturing gel load 180 300 ng of amplified DNA per well usually 5 10 of a 100 l PCR volume from a 100 ng DNA template A wild type control should be run on every gel 4 Add an equal volume of 2x gel lo...

Страница 21: ...are recommended These two different perpendicular gel formats consist of a set of spacers that provide casting at the side of the gel sandwich via the stopcock To insure proper alignment make sure all...

Страница 22: ...y 5 Place the sandwich assembly in the alignment slot the slot without cams of the casting stand with the short glass plate forward Figure 4 7 Loosen the sandwich clamps and insert an alignment card t...

Страница 23: ...h until it fits into the middle notch on the comb Straighten the spacer and the comb The bottom of the middle spacer should also be flush against the glass plates Figure 4 8 Note The proper comb for a...

Страница 24: ...plates are off set one or both of the sandwich clamps may not be tightened Repeat steps 5 13 14 Tighten the comb gasket screws an additional one turn If it is tightened more the glass plates may crack...

Страница 25: ...me adjustment screw Place the volume setting indicator located on the syringe holder to the desired volume setting Tighten the volume adjustment screw For 7 5 x 10 cm gels 1 mm thick set the volume se...

Страница 26: ...ense the gel solution out of the syringe 10 Slide the tubing from the low density syringe to one end of the Y fitting Do the same for the high density syringe 11 Connect the 9 cm tubing with the luer...

Страница 27: ...ectrophoresis Assembling the Parallel Gradient Gel Sandwich For parallel gel formats a 16 x 16 cm gel sandwich size is recommended The parallel gel format does not require special casting grooves in t...

Страница 28: ...t card to keep the spacers parallel to the clamps Note Always use the alignment slot and alignment card to set the spacers in place Failure to use these can result in gel leakage while casting as well...

Страница 29: ...proper alignment with the lever on the gradient deliv ery system Slide each syringe into a syringe sleeve Move the sleeve to the middle of the syringe keeping the volume gradations visible Make sure...

Страница 30: ...from the HI syringe by turning the syringe upside down plunger cap towards the bench and gently tapping the syringe Push the solution to the end of the tubing Do not push it out of the tubing as loss...

Страница 31: ...a fluorescent ruler along the axis of the denaturant gradient when taking a photograph Then determine the distance along the gradient where the maximum split is seen between bands In the example in F...

Страница 32: ...d running CDGE gels are included in the DCode electrophoresis reagent kit for DGGE CDGE catalog number 170 9032 For different percent crosslinking use the equation below to determine the amount of Bis...

Страница 33: ...brown bottle for approximately 1 month A 100 denaturant solution requires re dissolving after storage Place the bottle in a warm bath and stir for faster results To cast constant denaturing gradient g...

Страница 34: ...n 1 It is important to optimize the PCR reaction to minimize unwanted products which may interfere with gel analysis The PCR products should be evaluated for purity by agarose gel electrophoresis befo...

Страница 35: ...perature Set the temperature ramp rate to 200 C hr to allow the buffer to reach the desired temperature the quickest 4 Preheat the buffer to the set temperature It can take 1 to 1 5 hours for the syst...

Страница 36: ...4 18 Attaching the clamps to the glass plate assembly 5 Place the sandwich assembly in the alignment slot the slot without cams of the casting stand with the short glass plate forward Figure 4 19 Loos...

Страница 37: ...ten the clamp screws until it is finger tight Casting CDGE Gels 1 Place the gray sponge onto the front casting slot The camshafts on the casting stand should have the handles pointing up and pulled ou...

Страница 38: ...a chemical denaturing gradient Amplified mutant and wild type DNA from the gene of interest is loaded onto a polyacrylamide gel containing a constant concentration of urea During electrophoresis the...

Страница 39: ...ing urea to the gel A denaturing urea gel will lower the theoretical melting temperature of DNA by 2 C for every mole of urea 32 33 In Figure 4 22 the theoretical melting temperature range on the DNA...

Страница 40: ...ermine the amount of Bis to add The example stock solution below is for an acrylamide bis ratio of 37 5 1 40 Acrylamide Bis 37 5 1 Reagent Amount Acrylamide 38 93 g Bis acrylamide 1 07 g dH2 O to 100...

Страница 41: ...g per 40 ml Adjust this amount for other concentrations of running buffer 50x TAE Buffer Reagent Amount Final Concentration Tris base 242 0 g 2 M Acetic acid glacial 57 1 ml 1 M 0 5 M EDTA pH 8 0 100...

Страница 42: ...rature and the temperature ramp rate RR can be adjusted by using the raise and lower buttons The C RR button is used to scroll between the two parameters Fig 4 23 The temperature controller displays t...

Страница 43: ...r rectangular plate 2 Place the short glass plate on top of the spacers so that it is flush with the bottom edge of the long plate 3 Loosen the black thumb screw of each sandwich clamp by turning it c...

Страница 44: ...alignment card Remove the sandwich assembly from the casting stand and check that the plates and spacers are flush at the bottom If they are not flush realign the sandwich and spacers to obtain a good...

Страница 45: ...oduplex molecules Heteroduplex molecules with as little as one mismatch can show a difference in mobility in a gel than homoduplex molecules Heteroduplexes are generated in the following ways during P...

Страница 46: ...sample being analyzed on the DCode system Therefore a 40 stock solution containing acrylamide and bis acrylamide bis should be made or a 2x DEM solution Reagents for casting and running a heteroduple...

Страница 47: ...e see note TEMED 40 l 40 l 10 Ammonium persulfate 400 l 400 l Total volume 40 ml 40 ml Add dH2 O to 40 ml and mix Cast the gel immediately after adding the TEMED and ammonium persulfate Note For 0 5x...

Страница 48: ...t Final Concentration 40 Acrylamide PDA see note 10 0 ml 10 10x TTE 2 0 ml 0 5x Formamide 6 0 ml 15 Ethylene Glycol 4 0 ml 10 dH2 O 17 6 ml TEMED 40 0 l 10 Ammonium persulfate 400 0 l Total volume 40...

Страница 49: ...maintains the desired buffer temperature and controls the temperature ramp rate in the DCode system Figure 5 1 The actual and set buffer temperatures are displayed in degrees Celsius The set temperat...

Страница 50: ...f the larger rectangular plate 2 Place a short glass plate on top of the spacers so that it is flush with the bottom edge of the long plate 3 Loosen the single screw of each sandwich clamp by turning...

Страница 51: ...the sandwich assembly from the casting stand and check that the plates and spacers are flush at the bottom If they are not flush realign the sandwich and spacers for a good seal Repeat steps 5 7 8 Wh...

Страница 52: ...Differences in mobility of the single strands between the control wild type DNA and the other samples indicate a mutation SSCP is a widely used mutation screening method because of its simplicity How...

Страница 53: ...stem can control the buffer temperatures between 5 25 C The electrophoresis cooling tank is outfitted with two cooling fingers Tygon tubing connects the cooling fingers in the electrophoresis tank to...

Страница 54: ...ith reference to two characteristics 1 The total monomer concentration T T g acrylamide g bis acrylamide x 100 Total Volume 2 The crosslinking monomer concentration C C g bis acrylamide x 100 g acryla...

Страница 55: ...Use the calculation for B determined above B gel final volume ml of 2 bis acrylamide D 2 bis acrylamide solution Acrylamide Bis Solutions 1x TBE Reagent 8 Gel 37 5 1 X Gel 40 Acrylamide 7 78 ml C from...

Страница 56: ...ng dye For extra control the undenatured samples can be run on the gel 3 Denature the samples at 95 C for 5 minutes and then place on ice 6 5 Temperature Controller The temperature controller maintain...

Страница 57: ...mperature control module onto the electrophoresis tank The clear loading lid should be on the temperature control module Note For electrophoresis runs between 20 25 C the chiller should be set to 5 to...

Страница 58: ...5 Loosen the sandwich clamps and insert an alignment card to keep the spacers parallel to the clamps Note Always use the alignment slot and alignment card to set the spacers in place Failure to use th...

Страница 59: ...camshafts on the casting stand should have the handles pointing up and pulled out Place the sandwich assembly on the sponge with the shorter plate facing forward When the sandwich is placed correctly...

Страница 60: ...DNA The third step amplifies the sequence of interest and incorporates a tailed primer sequence This tailed primer contains a T7 promoter and eukaryotic translation initiation sequence These sequences...

Страница 61: ...through a 0 45 filter and store at 4 C Polyacrylamide gels are described with reference to two characteristics 1 The total monomer concentration T 2 The crosslinking monomer concentration C T gm acryl...

Страница 62: ...he specified percent of gel X 4 Stacking Gel 0 125 M Tris pH 6 8 Reagent Amount 40 Acrylamide Bis 1 0 ml 0 5 M Tris HCl pH 6 8 2 5 ml 10 SDS 0 1 ml dH2 O 6 4 ml 10 Ammonium Persulfate 50 0 l TEMED 10...

Страница 63: ...e use This solution is good for 1 day only 2 Heat the samples at 95 100 C for 5 minutes 7 5 Temperature Controller The temperature controller maintains the desired buffer temperature and controls the...

Страница 64: ...Assemble the gel sandwich on a clean surface Lay the large rectangular plate down first then place the left and right spacers of equal thickness along the short edges of the larger rectangular plate...

Страница 65: ...to set the spacers in place Failure to use these can result in gel leakage when casting as well as buffer leakage during the run 6 Align the plates and spacers by simultaneously pushing inward on bot...

Страница 66: ...the comb 4 Into a 50 ml tube add the required amount of solution for casting the lower or separating gel Section 7 2 Add a final concentration of 0 09 v v each of ammonium persulfate and TEMED solutio...

Страница 67: ...rmanent marker to mark the wells 3 With the short glass plate facing the core position the gel sandwich so that the locating pins on the core are fitted into the grooves on the outside surface of the...

Страница 68: ...point check the integrity of the upper buffer seal If the buffer appears to be leaking pour the running buffer into a beaker remove the gel sandwich assemblies Section 8 4 re lubricate the gasket and...

Страница 69: ...ibrate to the lower ramp rate for 5 10 minutes before loading samples Press the C RR button to return to the temperature readout Heteroduplex CSGE and PTT Gels 1 The electrophoresis tank should contai...

Страница 70: ...blue and Xylene cyanol light blue Optional When using radioactive samples the pump may be turned off during electrophoresis to reduce radioactive contamination 3 Apply power to the DCode system and be...

Страница 71: ...the top of the core b For 16 x 16 cm and 16 x 20 cm gels remove the sandwich assembly with your index fingers below the sandwich clamps and your thumbs resting on the latches on the core Gently remov...

Страница 72: ...ioisotopes must be autoradiographed or exposed to a storage phosphor imaging screen GS 525 Molecular Imager screen Carefully place a 3MM Whatman paper on top of the gel Gently slide your hand across t...

Страница 73: ...DCode system 9 1 Equipment Problem Cause Solution Controller No display with power on Burned out fuse Replace fuse located near power cord connection Buffer not circulating Buffer level too low Add b...

Страница 74: ...ss plate Wrong comb gasket Make sure correct comb gasket is used Misaligned comb gasket Insure that comb gasket notches are against spacer notches Misaligned plates Pressure clamp may force plates to...

Страница 75: ...NA unresolved run recommended 2 Recalculate gradient range from perpen dicular gel or run a time course gel Air bubbles in gel Clean glass plates Fuzzy DNA bands Clean wells before use Check for match...

Страница 76: ...tes 4 Do not overload sample well Reduce sample volume Skewed or distorted bands 1 Impurities in acrylamide Filter before use or DNA spikes in gel Check shelf life date of acrylamide solution 2 Carefu...

Страница 77: ...s Bands did not migrate far 1 Increase run time enough into gel 2 Decrease acrylamide concentration 3 Increase voltage DNA leaks between wells 1 Acrylamide not polymerized Add more TEMED and ammonium...

Страница 78: ...elf life date of acrylamide 2 Carefully load DNA in wells Do not pierce or puncture wells PTT Smile effect band pattern curves Decrease power setting or fill lower chamber upward at both sides of gel...

Страница 79: ...6 x 16 cm max two per run 16 x 10 cm max two per run 7 5 x 10 cm max four per run Spacers available 0 75 1 0 and 1 5 mm Combs 16 well comb compatible with 8 well multi channel pipettor 20 well comb 25...

Страница 80: ...Fischer S Hurley I Silverstein K and Lumelsky N Ann Rev Biophys Bioeng 13 399 423 1984 13 Hovig E Smith Sorensen B Uitterlinden A and Borresen A Pharmacogenetics 2 317 328 1992 14 Yoshino K Nishigaki...

Страница 81: ...DCode System for CDGE 120 V includes electrophoresis temperature control module sandwich core CDGE kit for 16 cm gel casting 2 sets of 16 cm plates 2 sets of 1 mm spacers two 20 well 1 mm combs contr...

Страница 82: ...tings required for gradient gel casting 170 9126 DGGE Kit 10 cm includes 2 sets of 10 cm plates 2 sets of 1 mm spacers two 2 well 1 mm prep combs sandwich clamps pressure clamp comb gasket and holder...

Страница 83: ...fate 170 9171 DCode Electrophoresis Reagent Kit TTGE includes 500 ml 40 acrylamide bis solution 37 5 1 1 kg urea 2 x 1 liter 50x TAE buffer 10 ml of 10mg ml EtBr 1 ml of 2x Gel loading dye 5 ml TEMED...

Страница 84: ...80...

Страница 85: ...io Rad Laboratories Ltd 14 Homa Street P O Box 5044 Rishon Le Zion 75150 Phone 03 951 4124 Fax 03 951 4129 Italy Bio Rad Laboratories S r l Via M Peroglio 23 00144 Rome Phone 34 91 590 5200 Fax 34 91...

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