LI-COR 4300 Manual
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LI-COR 4300 Manual

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Microsatellite
Analysis
Manual
Model 4300
DNA Analyzer
®

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Summary of Contents for LI-COR 4300

  • Page 1 Microsatellite Analysis Manual Model 4300 DNA Analyzer ®...

  • Page 3: Table Of Contents

    Table of Contents Section 1. Plate Organization Introduction....................1-1 The Hamilton 8-Channel Syringe..............1-1 Microplate Configurations ................1-1 Section 2. Gel Preparation and Electrophoresis Choosing Plates and Spacers .................2-1 Choosing a Gel....................2-1 Plate Assembly ....................2-2 Plus Gel Preparation...................2-6 Preparing Gel Solutions From Other Manufacturers ........2-7 Plus Pouring a 6.5% KB Gel................2-10...
  • Page 4 Section 4. Appendices Calculation ....................4-1 Calculation of Oligonucleotide Concentration (nmol) Given Optical Density (O.D.)................... 4-2 Protocol for 96-well Paper Combs ..............4-3...

  • Page 5: Section 1. Plate Organization

    The Hamilton 8-Channel Syringe The Hamilton 8-channel 0.2 mm syringe (Hamilton Co., Part #84511) has syringe needles are spaced 9 mm apart. Since the wells formed by LI-COR 64-well combs are on 2.25 mm centers, the 8-channel syringe can be used to load every fourth well of 0.25 mm gels.
  • Page 6 Section 1 are numbered according to the lane number that will be loaded. Both configurations satisfy Saga’s requirement of at least 5 size standard lanes for di- nucleotide repeats (7 lanes or more are better), as should any plate configurations you design. The first example shows how to load half of a full microplate on one gel and the other half on a second gel.
  • Page 7 Plate Organization Loading 60 Samples on a 64-well Comb Left syringe tip Figure 1-2. Microplate showing Load # starts at well #: 60 sample loading format. final load (a single lane) goes in lane 65 56 60...

  • Page 9: Section 2. Gel Preparation And Electrophoresis

    Fast Run Plate Length 25 cm 18 cm Spacer Thickness 0.25 mm 0.25 mm Plus Plus Gel Composition 6.5% LI-COR KB 6.5% LI-COR KB Run time (for 350 bases) 1.25 hours 45 minutes Reload gel after 1.5 hours 1 hour...

  • Page 10: Plate Assembly

    Section 2 Plate Assembly The following items are required to assemble the electrophoresis apparatus: • Gloves (non-powdered) • Safety glasses • Non-abrasive tissues (Kaydry and Kimwipes) • Front plate (notched) • Back plate (rectangular) • 1 set of spacers • Comb •...
  • Page 11 Gel Preparation and Electrophoresis Rinse the plates with deionized distilled water. Use a pump spray bottle to spray a fine mist of 70% ethanol/ddH O for a final rinse. Stand plates in a rack to air dry. Preparing Stock Bind Silane Solution Add 50 µl of bind silane to 10 ml of 100% ethanol.
  • Page 12 Section 2 Use a cotton swab to apply the solution on the inside of the short plate over the area below the edge of the notch where the wells will form (Figure 2-1). If you are using a squaretooth comb, apply solution to the rear plate using the front plate as a guide to determine where to place the bind silane onto the rear plate (other combs do not require back plate silane treatment).
  • Page 13 Gel Preparation and Electrophoresis Place the front plate on top of the rear plate (gel side down) and align the spacers with the outside edges of the plates. Make sure that the plates are aligned evenly at the bottom. Make sure the rails are completely dry from prior runs before assembly. Place the left and right rail assemblies over the plate edges.

  • Page 14: Kb Plus Gel Preparation

    Section 2 Tighten the glass clamp knob on each rail. Tighten only until finger tight (just past the point of resistance). Over tightening can break or dis- tort the glass plates. Over tightening is also one of the primary causes of “smiles”...

  • Page 15: Preparing Gel Solutions From Other Manufacturers

    Gel Preparation and Electrophoresis Plus Preparing KB Buffer Gel and running buffer solutions are prepared from a standard 10x TBE buffer. Plus Plus For best results, KB buffer is recommended for use with KB gel matrix. 10x TBE Buffer: Plus Empty the contents of the KB 10X TBE pouch into a 1 liter beaker.
  • Page 16 Section 2 Prepare 10x TBE as follows: Add the following to a 1000 ml beaker: Component Amount Molarity Tris Base 107.8 g 0.89 M Boric Acid 55.0 g 0.89 M EDTA (disodium salt) 7.4 g 0.02 M Distilled water 950 ml TOTAL VOLUME 1000 ml Stir to dissolve.
  • Page 17 20 ml A number of other commercial acrylamides can be used to cast gels for the LI-COR system including RapidGel-XL (USB) and Sequagel (National Diagnostics). To mix the gel solution in one beaker: Place a 100 ml beaker with a stir bar on a balance and tare the balance.

  • Page 18: Pouring A 6.5% Kb Plus Gel

    Section 2 Mixing the Gel Solution Mix well at room temperature and filter the solution using a membrane filtration system (syringe top or bottle top from any supplier). Add 150 µl of 10% APS per 20 ml of gel solution and swirl gently. Just before pouring, add 15 µl TEMED.
  • Page 19 Gel Preparation and Electrophoresis For 25 cm gels, a notch on the back of each rail that allows the appara- tus to rest on the uppermost metal posts on the casting stand (Figure 2-6). This slight incline improves the flow of the gel between the plates. Figure 2-6.
  • Page 20 Section 2 Insert the comb. Figures 2-8 and 2-9 show how to insert the mylar sharkstooth and rectangular tooth combs after pouring the gel. Instruc- tions for inserting paper combs are given in the Appendices (Section 5). The sharkstooth comb is inserted upside down during polymerization to make a trough which forms the base of the wells, and is then inverted before loading the samples.

  • Page 21: Pre-Electrophoresis Preparation

    Gel Preparation and Electrophoresis Place the casting plate into the grooved area in the rails normally occupied by the upper buffer tank. Tighten the two tank clamp knobs until finger tight. Alternatively, the upper buffer tank (with gasket) can be used in place of the casting plate. If you insert the upper buffer tank, be careful not to spill gel solution into the tank during gel pouring.
  • Page 22 Section 2 Remove the comb: Rectangular tooth comb: Carefully remove the comb by slowly pulling it straight out. This is a critical step, in that the well morphology must be maintained for sample loading. If the comb does not slide out easily, it may help to use a razor blade to score along the edge between the top of the comb and the back plate to break the gel seal.
  • Page 23 Gel Preparation and Electrophoresis Tighten the upper clamp knobs "finger tight". The electrophoresis appa- ratus is now fully assembled. Open the instrument door and place the lower buffer tank into position at the base of the heater plate. The tank has two recessed areas where the rails rest when the assembled gel apparatus is installed.
  • Page 24 Section 2 Place the upper and lower buffer tank lids onto the tanks. Insert the power cable on the upper buffer tank and connect it to the high voltage connector on the instrument chassis, as shown in Figure 5-14. Make sure that both connectors are fully inserted.
  • Page 25 Gel Preparation and Electrophoresis Place the upper and lower buffer tank lids onto the tanks. Insert the jumper on the upper buffer tank and connect it to the high voltage connector on the instrument chas- sis, as shown below. Make sure that both connectors are fully inserted. Upper buffer tank lid High voltage connector...

  • Page 26: Starting Runs

    • One of the accessories for the Model 4300 is a well visualization aid that can help you see rectangular wells. This aid has a mylar sheet that slides in behind the rear plate and can make the wells more visible.

  • Page 27: Disassembly

    Gel Preparation and Electrophoresis Disassembly Remove the buffer tank lids. For 18 cm gels, remove the entire gel apparatus from the instrument and carefully dispose of the buffer solution. For 25 cm or larger gels, the upper buffer tank has a fitting for draining the buffer solution, while the apparatus is still secured to the instrument.

  • Page 28: Cleanup

    Section 2 Cleanup After removing the upper and lower buffer tank lids and disconnecting the power cable, take the gel assembly off the sequencer and remove the upper buffer tank and rails. Remove the lower buffer tank and dispose of the buffer solution. Rinse the rails, spacers, and comb, and allow to air dry.
  • Page 29 Gel Preparation and Electrophoresis Pour distilled water onto the plate. Work the water around the plate with a gloved hand and watch for bubble formation from detergent res- idue. Repeat until no bubbles form. Repeat steps 3-5 above with the second plate. Rinse the plate with deionized distilled water.

  • Page 31: Section 3. Pcr Protocols

    PCR Protocols dNTP Recommendation We recommend using a dNTP mix containing 7-deaza-dGTP to minimize anomalies in migration that may cause difficulties in band sizing. Stock dNTP concentration is recommended to be 2 mM, whereas the concentration used in each reaction is 2 nmol for up to 6 loci per reaction. PCR Optimization As with any PCR, IRDye-labeled primer pairs may require optimization.

  • Page 32: Microsatellite Optimization

    Section 3 PCR reactions prior to loading the gel (pooling) or by including compatible primer pairs in the same PCR reaction (single tube). When selecting compatible primer pairs, the size range of the PCR products (smallest and largest alleles) must be such that loci are adequately spaced without overlap.
  • Page 33 PCR Protocols another may need 1.1 pmol to achieve the same band intensity. Even in monoplex reactions, a few primers may need 2-10 pmol of primer to achieve optimal results. These primers have relatively poor quality and should be redesigned if possible. To begin optimization, start the initial amplification with 0.1 to 0.5 pmol of primer for either monoplex or multiplex conditions.

  • Page 34: Lab Organization

    Section 3 Multiplex Reactions: For multiplexing, the "touchdown" protocol (54 °C High Temperature Enhanced) allows you to mix primer sets that vary widely in T "touchdown" protocol starts with a series of decreasing annealing temperatures for several cycles (usually decreasing1-2 °C per cycle for 5-10 cycles). These initial cycles allow primers with higher annealing temperatures to start amplifying before primers with lower melting temperatures.

  • Page 35: Preparing Genomic Dna

    PCR Protocols Preparing Genomic DNA DNA Quality Most DNA preps will work, but as with all PCR, the DNA should have a 260/ 280 ratio of 1.7 or greater. If the DNA was isolated from blood, the heme group can inhibit the DNA polymerase if not removed. DNA and Primer Amounts The amplification reactions can be approached in two ways, depending on the availability of DNA and the laboratory budget for primers.
  • Page 36 Section 3 The fragment sizes are listed below: Concentrated 50 - 350 Size 50 - 350 Size 50 - 700 Size Standard Standard Standard Size (bp) Size (bp) Size (bp) 500, 495 204, 200 204, 200 204, 200 105, 100 105, 100 Figure 3-2.

  • Page 37: Cycling Programs

    PCR Protocols Cycling Programs 1. Standard Step Temperature (°C) Time 5 minutes 20 seconds 20 seconds 30 seconds 3 minutes hold * Note: Annealing temperatures will alter depending on the T of the primers used. 2. 54 °C “Touchdown” PCR Step Temperature (°C) Time...
  • Page 38 Section 3 3. 47 °C “Touchdown” PCR Step Temperature (°C) Time 5 minutes 45 seconds 68 °C minus 2°C/cycle* 5 minutes 5 cycles total 1 minute 45 seconds 2 minutes 11 cycles total 1 minute 45 seconds 2 minutes 24 cycles total 1 minute 10 minutes hold...

  • Page 39: Tailed Primers

    Saga’s Locus Manager should be increased by the length of the primer incorporated in the PCR product. The M13 primer sequence can be used on both 800 and 700 channels. LI-COR M13 forward and reverse primer sequences are as follows:...
  • Page 40 Add 9 µl of the STR mixture from above to each tube or well and pipette gently to mix. If the thermocycler does not have a heated lid, add one drop of mineral oil to each well. Place the tubes or plate in the thermocycler. Start the cycling program. After completion of the program, add 2 µl of stop buffer to each tube or well and mix gently.

  • Page 41: Labeled Primers

    One of the STR primer pairs can be synthesized with a 5'-IRDye label. In this manner one strand of the resulting PCR product is labeled during amplification. The method is very robust and no purification is required prior to gel analysis. Custom primers are available from LI-COR. Protocol Enter the Standard program, or a custom program on the thermocycler (given earlier in this Section).
  • Page 42 3-12...

  • Page 43: Troubleshooting

    Troubleshooting Problem Cause Solution Multiplexing Not all loci amplify. Unamplified locus has lower Choose different locus. quality than others. Increase primer concentration. When using M13 tailed M13 labeled primer has Replace with fresh primer. primers, no loci amplify or a been exposed to light.
  • Page 44 Problem Cause Solution Bands in the 800 channel are IRDye 700 is more sensitive With the 800 image displayed, consistently weaker than the than IRDye 800. select Alter Intensity from 700 channel. the View menu in e-Seq and adjust the image until the bands are easier to see.
  • Page 45 Appendices Calculation There are two different formulas presented below that can estimate the melting temperature (T ) of a primer. The first is a very simple method (AT + GC)T that will give a rough estimate. This estimate is based on the following: 4 °C for every G or C and 2 °C for every A or T.

  • Page 46: Calculation Of Oligonucleotide Concentration (Nmol) Given Optical Density (O.d.)

    Section 4 Calculation of Oligonucleotide Concentra- tion (nmol) Given Optical Density (O.D.) Some DNA synthesis laboratories will include the O.D. and possibly the molecular weight (g/mol) with the primer that was synthesized. Others will calculate the concentration and report it in nmol. To calculate DNA concentration in nmol, both the O.D.

  • Page 47: Protocol For 96-Well Paper Combs

    Appendices Protocol for 96-well Paper Combs ® 96 well paper sharkstooth combs are used for microsatellite, AFLP , and other genotyping applications. These combs are capable of being inserted, loaded, and subsequently reloaded several times without removal. Protocol After pouring the gel, invert the 0.25 mm casting comb to create a trough that will form the bottom of each well.
  • Page 48 • P.O. Box 4000 • Lincoln, Nebraska 68504 USA Technical Support: 800-645-4260 North America: 800-645-4267 International: 402-467-0700 • FAX: 402-467-0819 LI-COR GmbH, Germany (Serving Europe and Africa): +49 (0) 6172 17 17 771 LI-COR UK Ltd., UK (Serving UK, Ireland, and Scandinavia): +44 (0) 1223 422104 www.licor.com...

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