DNA fragments generated with the KAPA Plant PCR Kits are A-tailed and suitable for use with TA cloning vectors.

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1 KAPA Plant PCR Kit Technical Data Sheet Product description Amplification of plant-derived DNA is a challenging application due to the diversity of plant tissue types and the potent PCR inhibitors contained in many of these. The KAPA Plant PCR Kit is optimized for the successful amplification of plant DNA from crude samples, DNA containing carry-over inhibitors from crude extraction methods, and purified DNA. The kit is capable of amplification from plant samples where conventional DNA amplification reagents either produce low yield or fail completely. The KAPA Plant PCR Kit is based on a novel DNA polymerase engineered via molecular evolution and exhibits improved tolerance to common plant-derived PCR inhibitors such as polyphenolics and polysaccharides. The unique characteristics of the enzyme result in robust amplification across a wide range of plant sample types, amplicon lengths, and crude extraction methods. The enzyme also provides faster extension rates than wild-type Taq polymerase without compromising performance. The enzyme formulation contains a small percentage of an engineered proofreading DNA polymerase, for even better performance. The enzymes are combined with proprietary antibodies that inactivate them until the first denaturation step. This eliminates spurious amplification products resulting from non-specific priming events during reaction setup and initiation, and increases overall reaction efficiency. DNA fragments generated with the KAPA Plant PCR Kits are A-tailed and suitable for use with TA cloning vectors. Applications The KAPA Plant PCR Kit is designed for the amplification of DNA fragments 5 kb in length and with a range of plant samples using a standardized reaction setup and cycling protocol. This allows the end-user to successfully amplify: from crude extractions of plant DNA containing carry-over inhibitors directly from leaf discs, crushed seeds, and other plant tissues from samples containing significant concentrations of plant-derived compounds samples where the available amount of starting DNA is limiting Storage and handling Long-term storage of kit components at -20 C is recommended. Kit components (BTS 1006 B) 250 U KAPA Plant PCR DNA Polymerase (2.5 U/µl) ml KAPA Plant PCR Buffer (2 ) containing MgCl 2 (1.5 mm final concentration) and dntps (0.2 mm each final concentration) ml MgCl 2 solution (25 mm) µl KAPA Plant PCR Enhancer (500 mm) ml KAPA Plant Sample Dilution Buffer (1 ) Version

2 Reaction setup A standard reaction setup is given below: Component Final concentration Volume in a 50 µl reaction* PCR water - Make up to 50 µl KAPA Plant PCR Buffer (2 ) (Contains MgCl 2 and 1 25 µl dntps) Forward primer (10 µm) 0.3 µm 1.5 µl Reverse primer (10 µm) 0.3 µm 1.5 µl KAPA Plant PCR enzyme (2.5 units/µl) 1 unit**/50 µl reaction 0.4 µl Template (DNA or crude sample) As needed As needed *Many sample types will work in a reaction volume of smaller than 50 µl, but this volume is recommended to start with, or for demanding sample types. **Some sample types may require the use of more than 1 unit of enzyme per 50 µl reaction. Use up to 5 units per 50 µl reaction for recalcitrant samples (also see Recommendations and guidelines for crude sample PCR). Notes: Ensure that all components are completely thawed and mixed properly before use. Briefly centrifuge tubes before opening. Note that the KAPA Plant PCR Buffer is somewhat viscous; it is therefore recommended that the water is added first when setting up the PCR master mix, then the KAPA Plant PCR Buffer. To ensure that all the buffer has been added, flush the pipette tip by pipetting up and down a couple of times. The KAPA Plant PCR Buffer contains MgCl 2 at a final concentration of 1.5 mm. Separate MgCl 2 (25 mm) is supplied with the kit for use with assays that require additional optimization. A final concentration of 1.5 mm MgCl 2 is usually sufficient for extracted or purified DNA, whereas 2.0 mm MgCl 2 is recommended for crude samples, or experiments that employ various sample types. KAPA Plant PCR Enhancer is supplied with the kit and may be included in the reaction at 1 5 mm final concentration ( µl/50 µl). KAPA Plant PCR Enhancer is recommended for difficult assays that do not improve after MgCl 2 and enzyme concentration have been optimized. KAPA Plant Sample Dilution Buffer setup: Add a leaf disc of approximately 1.2 mm diameter (or similar-sized other sample) to µl dilution buffer in a PCR tube, crush the sample with a pipette tip*, vortex, centrifuge, and use µl of the supernatant for PCR. Samples may be stored in dilution buffer for a limited time at 4 ºC or for longer periods at -20 ºC. The stability of different plant samples will vary and should be tested by the user. For maximum storage life, it is strongly recommended that the sample is centrifuged and the supernatant transferred to a new tube for storage at -20 ºC. * A useful tool for this can be made by briefly flaming a 100 µl pipette tip to seal it. The sealed end of the pipette tip will usually have a diameter very suitable for crushing samples at the bottom of a PCR tube (use a twisting motion for best results). Version

3 Cycling parameters Step Temperature ( C) Time No. of cycles Initial denaturation min, depending on 1 sample type 1 Denaturation s Annealing 4, s Extension s/kb Final extension s/kb 1 Hold For crude samples, use 10 min initial denaturation. For purified DNA, use 3 min initial denaturation. 2. Use at least 20 s denaturation time for 50 µl reaction volumes. 3. For crude samples, 40 cycles is recommended as a starting point. Increase or decrease cycle number according to results obtained (see Recommendations and guidelines for crude sample PCR). 4. Use the average primer T m + 5 ºC to start. Performing an annealing temperature gradient PCR is recommended for further optimization. Start with 15 sec annealing time; the annealing time may be reduced to 10 sec if non-specific amplification is observed which cannot be fixed by increasing the annealing temperature. 5. For primers with optimal annealing temperatures of 68 C or higher, a two-step cycling profile with annealing/extension at C may be used. 6. Start with 30 sec/kb extension time. If non-specific products of greater length than the desired PCR product are problematic, reduce the extension time to sec/kb. Recommendations and guidelines for crude sample PCR Successful DNA amplification from crude samples is dependent on determining a balance between the amount of template (i.e. amount of sample) and the concentration of PCR inhibitors in the sample. Amplification from crude plant tissue tends to improve as the amount of the tissue sample in the reaction is reduced. Increasing the reaction volume (e.g. 50 µl) and/or enzyme concentration is also recommended for successful amplification of more challenging sample types. The Harris Uni-Core sampling tool is recommended for maintaining consistent amounts of plant tissue per reaction. The 0.5 mm diameter punch is recommended for most applications. It is recommended that leaf discs are placed directly into the liquid in the PCR tube, rather than adding the master mix to the leaf disc. Do not further crush or damage leaf discs after addition to the tube. For very small seeds, such as Arabidopsis or tobacco, first crush the seed against the bottom of the PCR tube and then add the master mix. For larger seeds, use the 0.5 mm Uni-Core sampling tool to remove as small a piece as is possible and then add it directly to the PCR tube containing the master mix. Seeds from certain species may require pre-treatment with a lysis reagent such as KAPA Express Extract. Version

4 Please note that the number of PCR cycles used for crude samples tends to be high (40 50 cycles are typical). This is due to the mostly very low copy number of starting template, the effect of PCR inhibitors etc. Optimal cycle numbers for specific sample types/sizes will have to be determined empirically. Include appropriate positive and negative control reactions. The determination of optimal annealing temperature by performing an annealing temperature gradient PCR is highly recommended. Perform the gradient up to at least 72 ºC, or to such temperature where the yield begins to decrease significantly. If a primer set exhibits a wide T a range, it is recommended that the highest T a that results in acceptable yield is used. The KAPA Plant PCR system is capable of amplification directly from 0.5 mm diameter grapevine leaf discs (and leaf discs from other challenging species) in a 50 µl reaction, using 1 unit of enzyme (although 2 units of enzyme are recommended for markedly improved yields) PCR cycles are recommended for these troublesome samples. Increasing the amount of enzyme generally improves yield at a given number of PCR cycles, or enables fewer cycles for a given yield. For amplification from inhibitor-contaminated DNA preparations, 0.5 units of enzyme in a 25 µl reaction is usually sufficient. If initial tests with crude samples fail, then increasing the number of PCR cycles, and/or magnesium concentration above 2.0 mm in 0.5 mm increments (up to at least 3.0 mm) is recommended. Increasing the enzyme concentration to 2 units/50 µl reaction, or up to 5 units/50 µl reaction in extreme cases, may also be required. Particularly challenging sample types may require crushing the tissue in KAPA Plant Sample Dilution Buffer (see Notes under Reaction Setup for protocol), or pretreatment with a lysis reagent such as KAPA Express Extract. In cases where addition of extra magnesium and/or enzyme does not improve results, KAPA Plant PCR Enhancer may be included at 1 5 mm final concentration. The addition of ß-mercaptoethanol (BME) to PCR reactions at % v/v, or dithiothreitol (DTT) at 1 5 mm final concentration may also improve results with certain crude samples. Troubleshooting Before attempting amplification from crude samples with a given primer set, first optimize the reaction conditions (especially annealing temperature) with purified DNA. Remember to always store and dilute DNA and primers in a buffered solution (ph ). The remedies suggested for the problems given below may first be tried individually, then in combination as appropriate: Low yield or no amplification at all Increase the magnesium concentration and/or increase the number of PCR cycles For purified DNA, increase the amount of template For a crude tissue sample, decrease the amount of sample in the reaction/increase the reaction volume Use the KAPA Plant Sample Dilution Buffer or KAPA Express Extract with the crude sample Increase the enzyme concentration Use KAPA Plant PCR Enhancer, BME or DTT in the reaction Decrease the annealing temperature Increase the extension time Version

5 Check primer sequences Non-specific amplification or smearing Increase the annealing temperature and/or decrease the annealing time Decrease the number of cycles Decrease the primer concentration Decrease the magnesium concentration, if > 1.5 mm was used Decrease the enzyme concentration, if > 1 unit/50 µl reaction was used Decrease the extension time Decrease the amount of template Redesign the primers Version

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