Buccal Swab DNA Extraction
Contents
1. BuccalQuick PCR Amplification Regular PCR Use 0 5 2ul of the final extract for a 25 50 ul PCR reaction Real time PCR Use 0 5 1ul of the final extract for a 25 50ul real time PCR reaction Using excess final extract may negatively affect the real time PCR reaction The PCR result may vary depending on the PCR reagents used For first time user we recommend performing a titration test Use 0 5ul tul and 2ul of the final extract as a template for a 25 50ul PCR reaction to determine the best sample volume for your PCR An optimized PCR master mix is available at TrimGen Master Mix Cat No GR048 TrimGen Corporation BuccalQuick Troubleshooting Guide Problem Suggestions OD ratio is below QC criteria can still use the extracted DNA for PCR Yes BuccalQuick is a homogenous extraction method The OD ratio is lower than column or bead extraction method The low OD ratio will not affect the PCR amplification ODseo is too high Use the Blank control a reagent control to calibrate the spectrophotometer and then measure the OD of sample DNA concentration calculated from the equation is low and subsequently PCR does not amplify properly Incomplete release of buccal DNA from the swab The swab type and conditions significantly affect DNA release In general a cotton swab or swab with fungi growth gives poor DNA release Soaking the swab for an increased period of time at 55 C2. Cultured cells Isolated white blood cells The cells need to be washed with PBS before the extraction TrimGen Corporation BuccalQuick Kit Contents BQ 50 Gomponent 50 extractions BQ Solution 18 ml Enzyme Mix 425 ul International customers are provided with lyophilized enzyme mix and dilution buffer Use 500 ul buffer to solubilize the enzyme mix CAUTION The reagents contain hazardous chemicals Wear suitable eye protection and gloves FIRST AID In case of skin contact flush with water In case of ingestion or eye contact seek medical attention Materials and Equipment Needed 2 ml screw cap tubes Laboratory incubator or water bath Heat Block Vortex Mixer TrimGen Corporation BuccalQuick Procedure Pre heat an incubator or water bath or heat block to 55 C and a heat block to 95 C 1 Collect 2 ml screw cap tube for each swab Label the tube with sample ID and place the tube in a tube rack Blank control Add an extra tube to Step 1 Label the tube as BC and process it as a sample without a swab This BC tube serves as the Blank Control for OD260 280 calibration 2 Prepare Extraction Buffer BQ Solution 300 pl x ul total sample Enzyme Mix 7 ul x X ul total sample For pipetting error Transfer above calculated volumes of both reagents to one tube and mix the contents by vortexing This is the Extraction Buffer 3 Transfer 300 ul of Extraction Buffe
3. TrimGen Corporation BuccalQuick BuccalQuick Buccal Cell DNA extraction User Manual V1 2 TrimGen Corporation BuccalQuick TrimGen Corporation BuccalQuick Table of Contents Introduction 5 Frequently Asked Questions 6 Kit Contents 7 Materials and Equipment Needed 7 Procedure 8 DNA Concentration Measurement 9 Troubleshooting Guide 10 Storage Upon receipt store the kit at 2 8 C Storage Information Storage options Shelf life Kits stored at 2 8 C 6 months Kits stored at 20 C 1 year TrimGen Corporation BuccalQuick Limited Product Warranty It is imperative that users strictly adhere to this manual Failure to do so will void TrimGen s guarantee of this product TrimGen Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose This product is designed for research use only not use for diagnostic purpose The purchaser must determine the suitability of the product for its particular use No claim or representation is intended for use of this product to identify any specific organism or for a specific clinical use diagnostic prognostic therapeutic or blood banking Notice to Purchaser The purchase of BuccalQuick products includes a limited nonexclusive license to use the kit This license does not grant rights to reproduce or modify the kit for resale or to use the BuccalQuic
4. k kit to manufacture commercial products without written approval of TrimGen Corporation No other license expressed implied or by estoppels is granted Product Safety and Liabilities When working with the kit reagents always wear a suitable lab coat disposable gloves and protective goggles TrimGen Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the misuse the results of use or the inability to use this product TrimGen Corporation BuccalQuick Introduction Buccal cells are a convenient source to collect genomic DNA for genetic analysis This kit is designed to efficiently extract genomic DNA from a buccal swab or brush The whole extraction process is performed in less than 10 minutes No binding wash elute and centrifugation steps is required The kit can directly extract the DNA from a form swab TrimGen s Easy Swab dried and stored in room temperature for 2 years after buccal cell collection FEATURES Simple Single tube few steps no centrifugation High Yield DNA extracted from one buccal swab is sufficient to perform up to 100 PCR reactions DNA yield may vary if using different types of swabs The result is obtained from TrimGen s Easy Swab catalog number ES 100 Time Savings Less than 10 minutes extraction time Non detergent Detergents will affect downstream application The extraction buffer of BuccalQuick does not co
5. ntain any type of detergent Application Buccal Cell DNA extraction Culture Cell DNA extraction TrimGen Corporation BuccalQuick Frequently Asked Questions Q I have a sample from a different type of swab can use the BuccalQuick to perform the DNA extraction A Yes You need to increase the soaking time in the Extraction Buffer The swab type significantly affects the DNA release In general the cotton swab has a poor DNA release TrimGen provides Easy Swab Cat No ES 100 specially designed for buccal DNA collection The Easy Swab can release more than 90 of collected DNA The DNA yield from one Easy Swab is sufficient for up to 100 PCR applications Q Do need to adjust my PCR conditions A No The extraction buffer of BuccalQuick does not contain any type of detergent The final extract can be directly used for PCR it is not necessary to adjust the conditions of PCR amplification Q How much of the final extract should be used for PCR A PCR conditions vary by laboratory The extraction buffer of BuccalQuick is optimized for most commercial PCR kits It uses 1 2 ul for a 50 ul PCR reaction or 0 5 1 ul for 25 50 ul real time PCR reactions For home brew PCR a titration of final extracts 0 5ul Tul 2ul Sul and 5ul will help to find out the best condition for your PCR system Q Can use the kit to extract DNA from other cell samples A Yes You can use the kit to extract the DNA from
6. r to each tube 4 Place the swab head into the Extraction Buffer in the tube and twist the swab vigorously 8 10 times No swab for blank control tube Before discarding the swab try to remove all the liquid remaining in the swab head by pressing the swab head against the tube wall then discard the swab Alternative Cut the swab head into the Extraction Buffer Keep the swab head in the buffer during extraction 5 Cap the tube and vortex for 10 seconds at high speed 8 TrimGen Corporation BuccalQuick 6 Incubate the tube at 55 C for 1 5min a longer incubation will help to increase the yield of DNA 7 Heat the tube at 95 C for 3 minutes 8 The sample is ready for PCR DNA Storage The extracted DNA can be stored at 20 C DNA Concentration Measurement Transfer 10 ul of the final extract to a new tube Add 40 ul of water to make a 1 5 dilution Use the diluted Blank Control to calibrate the OD260 and OD280 to zero then measure the OD of samples The following equation can be used to estimate the DNA concentration DNA Conc ng ul 62 9 x OD260 36 x OD2s0 x dilution factor 5 A convenient calculation form is available online http trimgen com products BQ DNA calculation xls DNA Quality BuccalQuick is a homogeneous extraction method The final extract includes DNA and proteins A typical ODzeo280 ratio is 0 6 1 5 The low OD ratio will not affect the PCR TrimGen Corporation
7. will help to release the DNA DNA concentration calculated from the equation is high but the PCR does not amplify properly DNA concentration is too high Dilute the final extract with water then perform the PCR The extraction buffer is optimized for most commercial PCR kits If you use a home brew PCR the conditions may be different To find out the best conditions for your PCR a titration of the final extract 0 5ul 1ul 3ul and 5ul will help you to find the best volume for PCR amplification 11 TrimGen Corporation BuccalQuick 12
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