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Plasmid DNA Extraction Miniprep Kit
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1. 40 mL MINI 6 Elution Solution 1 5mL 15 mL 30 mL RNase A 50mg ml 6uL 60 uL 120 uL MINI Column 10 pcs 100 pcs 200 pcs Collection Tube 10 pcs 100 pcs 200 pcs User Manual 1 1 1 Add 1 5 mL 15 mL 30 mL ethanol 96 100 to MINI 4 Washing 1 Solution before first use x Add 8 mL 80 mL 160 mL ethanol 96 100 to MINI 5 Washing 2 Solution before first use BIO BASIC Toll Free 1 800 313 7224 Order www biobasic com 3 Plasmid DNA Extraction Miniprep Kit Important Notes 1 Solutions provided in this kit contain irritants wear gloves and lab coat when handling 2 Briefly spin RNase A tube to remove drops from the inside of the lid Add 0 1 mL of MINI 1 Resuspen sion Solution into RNase A tube and mix well Transfer the mixture into MINI 1 Resuspension Solution bottle and store at 4 C 3 Check MINI 2 Cell Lysis Solution before use Warm MINI 2 Cell Lysis Solution at 55 C for 10 mins if any recipitation formed Prevent vigorous shaking of the MINI 2 Cell Lysis Solution 4 To avoid acidification of MINI 2 Cell Lysis Solution from CO in the air close the bottle immediately after use 5 For 9K 006 0009s add 1 5 mL ethanol 96 100 to MINI 4 Washing 1 Solution before first use For 9K 006 0009 add 15 mL ethanol 96 100 to MINI 4 Washing 7 Solution before first use For 9K 006 0010 add 30 mL ethanol 96 100 to MINI 4 Washing 7 Solution before first use 6 For 9K 006 0009s add 8 mL ethanol 96 100 to MI
2. genomic DNA e Continue inverting the tube until the lysate becomes clear Do not let the mixture incubate for more than 5 min 5 Add 350 uL of MINI 3 Neutralization Solution and invert the tube 5 times immediately but gently e Invert immediately after adding MINI 3 Neutralization Solution will avoid localised precipitation e Do not vortex at this step as it may shear genomic DNA 6 Centrifuge for 10 min During centrifuging place a MINI Column in a Collection Tube 7 Transfer the supernatant carefully to MINI Column Centrifuge for 1min then discard the flow through e Do not transfer any white pellet into the column 8 Add 400 uL of MINI 4 Washing 1 Solutionto MINI Column Centrifuge for 1min then discard the flow through e Make sure that ethanol 96 100 has been added into MINI 4 Washing 1 Solution 9 Add 750 uL of MINI 5 Washing 2 Solution to MINI Column Centrifuge for 1min then discard the flow through e Make sure that ethanol 96 100 has been added into MINI 5 Washing 2 Solution 10 Centrifuge for an additional 3 min to dry the column e Important This step will remove any residual liquid that could inhibit subsequent enzymatic reaction 11 Place MINI Column to a new 1 5 mL microcentrifuge tube not provided 12 Add 50 uL 100 uL of MINI 6 Elution Solution or ddH20 PH 7 0 8 5 to the center of MINI Column membrane Stand the column for 1 min Make sure that the MINI 6 Elution Solution is dispensed on the ce
3. Ei BIO BASIC Plasmid DNA Extraction Miniprep Kit fs X IN 9K 006 0009s 10 preps E 9K 006 0009 100 preps 9K 006 0010 200 preps For Research Use Only Index C Plasmid DNA Extraction Miniprep Kit Introduction 3 Important Notes 4 General Protocol 5 Troubleshooting 6 XN S BIO BASIC INC Plasmid DNA Extraction Miniprep Kit Introduction Bio Basic Plasmid DNA Extraction Miniprep Kit is an excellent tool offering a speed and economic method to purify plasmid DNA from bacteria cultures This technology is based on binding DNA to silica based membranes in chaotropic salts and washing DNA with specially formulated solutions Compared with other harmful and time consuming procedures such as phenol chloroform extraction and ethanol precipitation Bio Basic Plasmid DNA Extraction Miniprep Kit shortens the handling time to about 25 min The high quality plasmid DNA can be used directly for any downstream applications Specification Sampling Yield Handling time 1 5 mL overnight culture up to 20 ug for high copy plasmids about 25 min Kit Contents 9K 006 0009s 9K 006 00010 9K 006 00010 10 preps 100 preps 200 preps MINI 1 Resuspension Solution 3mL 30 mL 60 mL MINI 2 Cell Lysis Solution 3mL 30 mL 60 mL MINI 3 Neutralization Solution 4mL 40 mL 80 mL MINI 4 Washing 1 Solution concentrated 3 5mL 35 mL 70 mL MINI 5 Washing 2 Solution concentrated 2mL 20 mL
4. NI 5 Washing 2 Solution before first use For 9K 006 0009 add 80 mL ethanol 96 100 to MINI 5 Washing 2 Solution before first use For 9K 006 0010 add 160 mL ethanol 96 100 to MINI 5 Washing 2 Solution before first use 7 All centrifuge steps are performed at full speed 14 000 rpm or 10 000 x g in a microcentrifuge A Brief Procedure Bacterial Culture Resuspend MINI 1 Resuspension Solution Lyse MINI 2 Cell Lysis Solution centrifuge Netralize MINI 3 Neutralization Solution Bi ind centrifuge UY Wash MINI 4 Washing 1 Solution MINI 5 Washing 2 Solution centrifuge 7 Elute MINI 6 Elution Solution centrifuge eee J Pure Plasmid DNA J 4 Order www biobasic com Toll Free 1 800 313 7224 BIO BASIC Plasmid DNA Extraction Miniprep Kit General Protocol 1 Transfer 1 5ml of well grown bacteria culture to a microcentrifuge tube not provided 2 Spin down the bacteria by centrifuging for 1 2 min and discard the supernatant completely 3 Add 250 uL of MINI 1 Resuspension Solution to the pellet and resuspend the cells completely by pipetting or vortexing e Make sure that RNase A has been added into MINI 1 Resuspension Solution No cell pellet should be visible after resuspension of the cells 4 Add 250 uL of MINI 2 Cell Lysis Solution invert the tube 5 times to lyse the cells and incubate at room temperature for 2 min e Do not vortex at this step as it may shear
5. nter of the membrane and is absorbed completely 13 Centrifuge for 1 min to elute plasmid DNA 14 Store plasmid DNA at 4 C or 20 C BIO BASIC Toll Free 1 800 313 7224 Order www biobasic com 5 Plasmid DNA Extraction Miniprep Kit Troubleshooting Low yield Bacterial cells were not lysed completely Too many bacterial cells were used If using bacterial culture with an OD600 gt 10 separate it into three tubes After MINI 3 Neutralization Solution addition break up the precipitate by inverting to ensure higher yield Overgrown of bacterial cells e Incubation time should not exceed 16 hours Bacterial cells were insufficient Ensure that bacterial cells have grown to an expected amount OD600 gt 1 after incubation under suitable shaking conditions and temperature Incorrect DNA MINI 6 Elution Solution Step Ensure that MINI 6 Elution Solution was added and absorbed to the center of MINI Column Matrix Incomplete DNA elution If size of DNA plasmids is larger than 10kb use preheated MINI 6 Elution Solution 60 70 C on Step 12 to improve the elution efficiency Incorrect MINI 5 Washing 2 Solution e Ensure that ethanol was added to MINI 5 Washing 2 Solution prior to use Eluted DNA does not perform well Residual ethanol contaminants e After MINI 5 Washing 2 Solution Step dry MINI Column with additional centrifugation at top speed for 3 min or incubation at 60 C for 3 min Genomic DNA C
6. ontamination Lysate prepared improperly e Gently invert the tube after adding MINI 2 Cell Lysis Solution The incubation time should not exceed 5 min Do not use overgrown bacterial culture Order www biobasic com Toll Free 1 800 313 7224 BIO BASIC Plasmid DNA Extraction Miniprep Kit Troubleshooting RNA Contaminates Plasmid DNA Insufficiency of RNase A activity in MINI 1 Resuspension Solution because of long term storage Prior to use MINI 1 Resuspension Solution ensure that RNase A was added If RNase A added to MINI 1 Resuspension Solution Buffer is out of date add additional RNase A into MINI 1 Resuspension Solution to a concentration of 50 ug ml then store at 4 C Too many bacterial cells were used reduce sample volume Smearing or degradation of Plasmid DNA Nuclease contamination eif used host cells have high nuclease activity e g endA strains perform this optional wash step to remove residual nuclease e After DNA Binding Step add 400ul of MINI 4 Washing 1 Solution into MINI Column and incubate for 2 min at room temperature e Centrifuge at full speed 14 000 rpm or 10 000 xg for 30 seconds e Proceed to standard MINI 5 Washing 2 Solution Step Plasmid DNA is not adequate for enzymatic digestions Eluted plasmid DNA contains residual ethanol e Discard the flow through after washing with MINI 5 Washing 2 Solution and centrifuge for an additional 3min Denatured Plasmid DNA migrate faste
7. r than supercoiled form during electrophoresis Incubation in MINI 2 Cell Lysis Solution too long Do not incubate longer than 5 min in MINI 2 Cell Lysis Solution BIO BASIC Toll Free 1 800 313 7224 Order www biobasic com 7 BIO BASIC Worldwide r For more information on pricing complete product line or to locate a Point of Sales near you please visit our web site or contact one of our Customer Service Representatives a order biobasic com Phone 1 905 474 4493 Toll Free Q 1 800 313 7224 1 905 474 5794 63 BIO BASIC INC Email Fax
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