FavorPrep Plasmid Extraction Mini Kit
Contents
1. User Manual FavorPrep Plasmid Extraction Mini Kit Cat No FAPDE 000 Mini 4 preps these FAPDE 001 100 preps FAPDE 001 1 300 preps For Research Use Only Kit Contents Brief procedure FAPDE 000 Mini FAPDE 001 FAPDE 001 1 4 preps_sample 100 preps 300 preps FAPD1 Buffer FAPD2 Buffer FAPD3 Buffer W1 Buffer concentrate Wash Buffer concentrate Elution Buffer FAPD Column Collection Tube RNase A Lyophilized User Manual Preparation of W1 Buffer and Wash Buffer by adding ethanol 96 100 Ethanol volume for W1 Buffer Ethanol volume for Wash Buffer 4ml Well grown bacterial culture l Harvest bacterial cells eResuspend FAPD1 Buffer eLyse FAPD2 Buffer e Neutralize FAPD3 Buffer f Centrifuge e Clarify the lysate by 18 000 x g 10 min centrifugation l SS m Centrifuge gt e Binding of plasmid 11 000 x g 30 sec iein Ei e Washing W1 Buffer Wash Buffer Centrifuge 11 000 x g 30 sec Centrifuge 18 000 x 9 3 min Drying column matrix Specification Principle mini spin column silica matrix Sample size L 5 mi Size of plasmid or construct lt 15 kb Centrifuge a Elution Elution Buffer 18 000 x g 1 min Operation time lt 25 minutes Typical Yield 25 40 ug Binding capacity 60 ug column Column applicability centrifugation and vaccum e Pure plasmid Important Notes 1 Store RNase A at 20 C upon2. immediately to neutralize the lysate e Invert immediately after adding FAPD3 Buffer will avoid asymmetric precipitation 6 Centrifuge at full speed 18 000 x g for 10 min to clarify the lysate During centrifugation place a FAPD Column in a Collection Tube 7 Transfer the suspernatant carefully to the FAPD Column and centrifuge at 11 000 x g for 30 seconds Discard the flow through and place the column back to the Collection Tube e Do not transfer any white pellet into the column 8 Add 400 ul of W1 Buffer to the FAPD Column and centrifuge at 11 000 x g for 30 seconds Discard the flow through and place the column back to the Collection Tube e Make sure that ethanol 96 100 has been added into W1 Buffer when first Use v 1014 9 Add 700 ul of Wash Buffer to the FAPD Column and centrifuge at 11 000 x g for 30 seconds Discard the flow through and place the column back to the Collection Tube e Make sure that ethanol 96 100 has been added into Wash Buffer when first use 10 Centrifuge at full speed 18 000 x g for an additional 3 minutes to dry the FAPD Column e Important step The residual liquid should be removed thoroughly on this step 11 Place the FAPD Column to a new 1 5 ml microcentrifuge tube not provided 12 Add 50 ul 100 ul of Elution Buffer or ddH20 to the membrane center of the FAPD Column Stand the column for 1 minute e Important step For effective elution make sure that the elution solution is dispensed on
3. prepared improperly e Gently invert the tube after adding the FAPD2 Buffer And the incubation time should not longer than 5 minutes Do Not use overgrown bacterial culture RNA Contaminates Plasmid DNA Insufficiency of RNase A activity in FAPD1 Buffer because of long term storage e Prior to using FAPD1 Buffer ensure that RNase A was added If RNase A added FAPD1 Buffer is out of date add additional RNase A into FAPD1 Buffer to a concentration of 50 ug ml then store 4 C eToo many bacterial cells were used reduce sample volume Smearing or degrading of Plasmid DNA Nuclease contamination elf used host cells have high nuclease activity g enA strains perform the following optional Wash Step to remove residuary nuclease a After DNA Binding Step add 400 ul of W1 Buffer into the FAPD Column and incubate for 2 minutes at room temperature b Centrifuge at full soeed 18 000 xg for 30 seconds cC Proceed to step 9 Plasmid DNA is not adequate for enzymatic digestions Eluted plasmid DNA contains residual ethanol e Make sure you have discarded the flow through after washing with Wash Buffer Step 9 and centrifuged for an addition 3 minutes Step 10 Denatured Plasmid DNA migrate faster than supercoilded form during electrophoresis Incubation in FAPD2 Buffer too long Do not incubate the sample longer than 5 minute in FAPD2 Buffer
4. recipit of kit 2 Add 0 5 ml of FAPD1 Buffer to a RNase A tube Dissolve the RNase A by vortexing Briefly spin the tube and transfer the total RNase A mixture back to the FAPD1 bottle mix well by vortexing and store the FAPD1 buffer at 4 C 3 If precipitates have formed in FAPD2 Buffer warm the buffer in 37 C waterbath to dissolve precipitates 4 Preparation of W1 Buffer and Wash Buffer by adding 96 100 ethanol not provided for first use 5 Centrifugation steps are done by a microcentrifuge capable of the soeed at 11 000 1 8000 x g General Protocol Please Read Important Notes Before Starting Following Steps 1 Transfer 1 5 ml of well grown bacterial culture to a centrifuge tube not provided 2 Centrifuge the tube at 11 000 x g for 1 minute to pellet the cells and discard the supernatant completely 3 Add 250 ul of FAPD1 Buffer RNase A added to the cell pellet and resuspend the cells completely by pipetting e Make sure that RNase A has been added into FAPD1 Buffer when first use e No cell pellet should be visible after resuspension of the cells 4 Add 250 ul of FAPD2 Buffer and gently invert the tube 5 10 times Incubate the sample mixture at room temperature for 2 5 minutes to lyse the cells e Do not vortex vortex may shear genomic DNA If necessary continue inverting the tube until the lysate become clear e Do not proceed the incubation over 5 minutes 5 Add 350 ul of FAPD3 Buffer and invert the tube 5 10 times
5. the membrane center and is absorbed completely e Note Do not Elute the DNA using less than suggested volume 50ul It will lower the final yield 7 13 Centrifuge at full speed 18 000 x g for 1 minute to elute plasmid DNA and store the DNA at 20 C Troubleshooting Low yield Bacterial cells were not lysed completely eToo many bacterial cells were used OD600 gt 10 Separate the bacterial culture into multiple tubes e After FAPD3 Buffer addition break up the precipitate by inverting to ensure higher yield Overgrown of bacterial cells elIncubation time should not longer than 16 hours Bacterial cells were insufficient eEnsure that bacterial cells have grown to an expected amount OD600 gt 1 after incubation under suitable shaking modes Incorrect DNA elution step eEnsure that Elution Buffer was added and absorbed to the center of the FAPD Column matrix Incomplete DNA Elution elf size of DNA fragments is larger than 10 kb use preheated Elution Buffer 60 70 C on slution step to improve the elution efficiency Incorrect preparation of W1 Buffer and Wash Buffer eEnsure that the correct volume of ethanol 96 100 was added to W1 Buffer and Wash Buffer pior to use Eluted DNA does not perform well Residual ethanol contamination e After Wash Step dry the FAPD Column with an additional centrifugation at top speed 18 000 x g for 5 minutes or incubation at 60 C for 5 minutes Genomic DNA Contaminates Lysate
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