User Manual FavorPrep Plasmid DNA Extraction Mini
Contents
1. than 5 minute in FAPD2 Buffer2. FavorPrep Plasmid DNA Extraction Mini Kit User Manual Cat No FAPDE 001 100 Preps FAPDE 001 1 300 Preps For Research Use Only v 1103 Introduction FavorPrep Plasmid Extraction Mini 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 washing DNA with ethanol contained Wash Buffer Compare with other harmful and time consuming procodure such as phenol chloroform extraction and ethanol precipitation FavorPrep Plasmid extraction kit shortens the handling time to about 25 minutes The high quality plasmid DNA can be used directly for the downstream application Specification Sampling 1 5 ml overnight culture Plasmid Size lt 12Kb Yield 20 30 ug of high copy plasmid Handing time about 25 min Kit Contents FAPDE 001 FAPDE 001 1 FAPD1 Buffer 30 ml 90 ml FAPD2 Buffer 30 ml 90 ml FAPD3 Buffer 40 ml 120 ml W1 Buffer concentration 35 ml 98 ml Wash Buffer concentration 20 ml 50 ml Elution Buffer 15 ml 35 ml RNase A 50mg ml 60 ul 180 ul FAPD Column 100 pcs 300 pcs Collection Tube 100 pcs 300 pcs User Manual 1 1 Add 13 ml 36 ml ethanol 96 100 to W1 Buffer when first open Add 80 ml 200 ml ethanol 96 100 to Wash Buffer when first open Important Notes 1 i Brief Procedure Buffer provided in this kit contain irritants Wear gloves and la
3. b coat when handling these buffer Brief spin RNase A tube to remove drops from the inside of the lid Add 1 ml of FAPD1 Buffer into RNase A tube and mix well Transfer the mixture into FAPD1 Buffer bottle and store at 4 C Check FAPD2 Buffer before use Warm FAPD2 Buffer at 55 C for 10 minutes if any precipitation formed Don t shake FAPD2 Buffer vigorously To avoid acidification of FAPD2 Buffer from CO2 in the air close the bottle immediately after use For FAPDE 001 add 13 ml ethanol 96 100 to W1 Buffer when first open For FAPDE 001 1 add 36 ml ethanol 96 100 to W1 Buffer when first open For FAPDE 001 add 80 ml ethanol 96 100 to Wash Buffer when first open For FAPDE 001 1 add 200 ml ethanol 96 100 to Wash Buffer when first open All centrifuge steps are done at full speed 14 000 rpm or 10 000 x g ina microcentrifuge well grown bacterial culture Resuspend FAPD1 Lyse FAPD2 centrifuge 4 U Neutralize FAPD3 g Bind centrifuge ka mi Wash w1 i Wash centrifuge 4 pa Elute Elution centrifuge 4 a Pure plasmid DNA General Protocol Transfer 1 5 ml of well grown bacteria culture to a microcentrifuge tube not provided Descend the bacteria by centrifuging for 1 2 min and discard the supernatant completely Add 250 ul of FAPD1 Buffer to the pellet and resuspend the cells completely by pipetting e Make sure that RNase A has been add
4. ed into FAPD1 Buffer when first open e No cell pellet should be visible after resuspension of the cells Add 250 ul of FAPD2 Buffer and genily invert the tube 5 times to lyse the cells and incubate at room temperature for 2 min e Do not vortex vortex may shear genomic DNA If necessary continue inverting the tube until the lysate beccome clear e Do not proceed this step over 5 min Add 350 ul of FAPD3 Buffer and invert the tube 5 times immediately but gently e Invert immediately after addind FAPD3 Buffer will avoid asymmetric precipitation Centrifuge for 10 min During centrifuging place a FAPD Column ina Collection Tube Transfer the suspernatant carefully to FAPD Column Centrifuge for 1 min then discard the flow through e Do not transfer any white pellet into the column Add 400 ul of W1 Buffer to FAPD Column Centrifuge for 1 min then discard the flow through e Make sure that ethanol 96 100 has been added into W1 Buffer when first open 9 Add 750 ul of Wash Buffer to FAPD Column Centrifuge for 1 min then discard the flow through e Make sure that ethanol 96 100 has been added into Wash Buffer when first open 10 Centrifuge for an additional 5 min to dry the column e Important step This step will remove the residual liquid completely that will inhibit subsequent enzymatic reaction 11 Place FAPD Column to a new 1 5 ml microcentrifuge tube not provided 12 Add 50 ul 100 ul of Elution Buffe
5. or incubation at 60 C for 5 minutes Genomic DNA Contaminates Lysate prepared improperly e Gently invert the tube after adding FAPD2 Buffer And the incubation time should not longer than 5 minutes Do Not use overgrown bacterial culture 5 Troubleshooting RNA Contaminates Plasmid DNA Insufficiency of RNase A activity in FAPD1 Buffer because of long term storage 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 50ug ml then store 4 C Too many bacterial cells were used reduce sample volume Smearing or degrading of Plasmid DNA Nuclease contamination If used host cells have high nuclease activity e g enA strains perform this Optional Wash Step to remove residuary nuclease e After DNA Binding Step add 400pl of W1 Buffer into FAPD column and column and incubate for 2 minutes at room temperature Centrifuge at full speed 14 000 rpm or 10 000 xg for 30 seconds e Followed using standard Wash Step Plasmid DNA is not adequate for enzymatic digestions Eluted plasmid DNA contains residual ethanol eMake sure you have discarded the flow through after washing with Wash Buffer Step 9 and centrifuged for an additional 3 minutes Step 10 Denatured Plasmid DNA migrate faster than supercoilded form during electrophoresis Incubation in FAPD2 Buffer is too long Do not incubate longer
6. r or ddH20 to the membrane center of FAPD Column Stand the column for 1 min e Important step For effective elution make sure that the elution solution is dispensed on the membrane center and is absorbed completely e Important Do not Elute the DNA using less than suggested volume 50ul It will lower the final yield 13 Centrifuge for 1 min to elute plasmid DNA 14 Store plasmid DNA at 4 C or 20 C Troubleshooting Low yield Bacterial cells were not lysed completely Too many bacterial cells were used ODs00 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 e Incubation time should not longer than 16 hours Bacterial cells were insufficient e Ensure that bacterial cells have grown to an expected amount ODs00 gt 1 after incubation under suitable shaking modes Incorrect DNA Elution Step e Ensure that Elution Buffer was added and absorbed to the center of FAPD Column Martix Incomplete DNA Elution elf size of DNA fragments is larger than 10 kb use preheated Elution Buffer 60 70 C on Elution Step to improve the elution efficiency Incorrect Wash Buffer Ensure that Ethanol was added to Wash Buffer pior to use Eluted DNA does not perform well Residual ethanol contamination e After Wash Step dry FAPD Column with additional centrifugation at top speed for 5 minutes
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