Contents - Biomiga
Contents
1. Elution Buffer 4 mL 20 mL 60 mL User Manual 1 1 1 Add 20 mL PD1468 00 or 96 mL PD1468 01 or 216 mL PD1468 02 96 100 ethanol to each DNA Wash Buffer bottle before use Safety Information Buffer C1 contains acetic acid wear gloves and protective eyewear when handling Buffer C1 contains chaotropic salts which may form reactive compounds when combines with bleach Do not add bleach or acidic solutions directly to the preparation waste Express Plasmid Midiprep Page 5 EZgene Express Plasmid Midiprep Protocol Inoculate 50 80 mL LB containing appropriate antibiotic with 100 ul fresh starter culture Grow at 37 C for 14 16 h with vigorous shaking Note The best way to prepare a starter culture Inoculate a single colony from a freshly grown selective plate into 1 ml LB medium containing the appropriate antibiotic and grow at 37 C for 6 8 h with vigorous shaking 250 rpm Warning Do not use more than 100 ml culture Need to scale up buffers if processing more than 100 mL culture Harvest the bacterial by centrifugation at 5 000 g for 10 min at room temperature Pour off the supernatant and blot the inverted tube on paper towels to remove residual medium Note Complete removal of residue medium is critical for bacteria lysis in the next step 3 Add 5 mL Buffer Al and completely resuspend bacterial pellet by vortexing or pipetting Ensure that R2. HEK293 restriction mapping library screening sequencing as well as gene therapy and genetic vaccinations Important Notes Plasmid Copy Numbers The yield of plasmid DNA depends on the origin of the replication and the size of the plasmid The protocols are optimized for high copy number plasmid purification For low copy number plasmids both the culture volume and the buffer volume need to be scaled up 2 times Please contact our customer service for further information and reference Table 1 for the commonly used plasmids Table 1 Commonly used plasmid and expected yield Plasmid Origin Copy Numbers Expected Yield ug per 50 mL pSC101 pSC101 5 5 pACYC PISA 10 12 5 10 pSuperCos pMB1 10 20 10 20 pBR322 pMB1 15 20 10 20 pGEM Muted pMB1 300 400 100 150 pBluescript ColE1 300 500 100 200 pUC Muted pMB1 500 700 150 250 Host Strains The strains used for propagating plasmid have significant influence on yield Host strains such as Top 10 and DH5a yield high quality plasmid DNA endA strains such as JM101 JM110 HB101 TG1 and their derivatives normally have low plasmid yield due to either endogenous endonucleases or high Page 2 Express Plasmid Midiprep Trouble Shooting Guide carbohydrates released during lysis We recommend transform plasmid to an endA strain if the yield is not satisfactory For purifying plasmid DNA from endA strains Table 2 we recommend use product PD171
3. 2 Table2 endA strains of E Coli EndA Strains of E Coli DH5a DHI DH21 JM106 M109 SK2267 SRB XLO TOP10 DH10B JM103 JM107 SK1590 MM294 Stbl2 as XL10 BJ5182 DH20 JM105 IM108 SK1592 Select96 stbiam AEE EndA Strains of E Coli C600 JM110 RRI ABLE C C3236 KW251 P2392 BL21 DE3 p 9 HB101 TGI TBI ABLE K PHI2S 1 392 pr7oo BL21 DE3 pLysS JMI01 IM83 TKBI HMS174 ES1301 M1061 Q358 BMH71 18 All NM strains All Y strains Problem Possible Reason Suggested Improvement Low Yield Poor Cell lysis e Resuspend pellet thoroughly by votexing and pipetting prior adding buffer B1 e Make fresh buffer B1 if the cap had not been closed tightly Buffer B1 0 2M NaOH and 1 SDS Low Yield Bacterial culture Grow bacterial 12 16 hours Spin overgrown or not down cultures and store the pellet at fresh 20 C if the culture is not purified the same day Do not store culture at 4 C over night Low Yield Low copy number Increase culture volume Increase the plasmid volume of buffer Al Bl Cl and 100 ethanol proportionally with the ratio of 1 1 1 2 1 2 No DNA Plasmid lost in Host Prepare fresh culture E coli Genomic DNA Over time incubation Do not vortex or mix aggressively contamination after adding buffer after adding buffer Bl Do not Bl incubate more t
4. Contents CONTENTS oeron oea EE E e EE EEEE EREE EEEO cece dea sae atin ees 1 Introductions leiert a a EE vei S A eS 2 Important Notes sinss se neend eaa Raa naa 2 Storage and Stability cece ccc e cee ce ence ee ene eee n cena ene ene een 3 Before Starting erans arer raai EEAS ceded andres AA APAA TARR 4 KitContehtS perseae rA a EA T EEA 5 Safety Information ccc cece cece eee ee ene ene ences eae eneeneenees 5 EZgene Plasmid Midiprep Spin Protocol cccccceeeeseeeeeee 6 Express Plasmid Midiprep Flow Chart ccceeceeceeeceeeseeeseeeeenee 8 Trouble Shooting Guide ccccccssecsccetceseceeeeeeteeeseesteeeneeeeeneeeaees 9 Express Plasmid Midiprep Page 1 Introduction Key to the kit is our proprietary DNA binding systems that allow the highly efficient binding of DNA to our ezBind matrix while proteins and other impurities are removed by Wash Buffer Nucleic acids are easily eluted with sterile water or Elution Buffer Unlike other kits in the markets no chaotropic salts are contained in the buffer of our patented plasmid purification kit The purified DNA is guanidine anion exchange resin residues free This kit is designed for fast and efficient purification of plasmid DNA from 50 to 80 mL of E coli culture The column has a plasmid DNA binding capacity of 1000 ug The purified DNA is ready for high performance of downstream applications such as transfection of robust cells such as
5. Nase A has been added into Buffer Al before use Note Complete resuspension is critical for optimal yield Add 5 mL Buffer B1 mix thoroughly by inverting 10 times with gentle shaking Incubate for 5 10 min to obtain a slightly clear lysate Complete lysis is critical for DNA yield The mixture of completely lysed bacteria looks transparent Attention Buffer B1 forms precipitation below room temperature if solution becomes cloudy warm up at 37 C to dissolve before use Add 6 mL Buffer C1 mix completely by inverting the tube 10 times and shaking for 5 times It is critical to mix the solution well if the mixture still appears conglobated brownish or viscous more mix is required to completely neutralize the solution Two options for clearing the lysates High speed centrifuge Transfer the lysate to a high speed centrifuge tube and centrifuge at 13 000 rpm 14 000 18 000 g for 15 min at room temperature Transfer the cleared lysate to a 50 ml conical tube Page 6 Express Plasmid Midiprep ezFilter syringe Pour the lysate into the barrel of the filter syringe and set the syringe ina 50 mL conical tube Incubate at room temperature for 10 min The white precipitates should float to the top Gently insert the plunger to expel the cleared lysate to the tube stop when feel strong resistance some of the lysate may remain in the flocculent precipitate Note To avoid clog of the syringe Use less than 100 mL of overnight cultu
6. from the date of purchase Express Plasmid Midiprep Page 3 Before Starting Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each step Important RNase A It is stable for more than half a year when stored at room temperature Spin down the RNase A vial briefly Add the RNase A solution to Buffer Al and mix well before use Store at 4 C Buffer B1 precipitates below room temperature It is critical to warm up the buffer at 50 C to dissolve the precipitates before use Buffer C1 may form precipitates below 10 C warm up at 37 C to dissolve the precipitates before use Keep the cap tightly closed for Buffer B1 after use Make sure the availability of centrifuge 13 000 rpm Especially after mixing the lysate with ethanol the sample needs to be processed immediately by centrifugation Carry out all centrifugations at room temperature For certain reasons Buffer KB are not involved in this kit from now on Materials supplied by users 100 ethanol High speed centrifuge 50 mL high speed centrifuge tubes 50 mL conical tubes Isopropanol if precipitate the plasmid DNA Page 4 Express Plasmid Midiprep Express Plasmid Midiprep Flow Chart Lysis and neutralization Clear Lysate through a Syringe filter Ear ig Bind DNA to the DNA column Wash the column with DNA Wash Buffer by a plunger Detach the column and i
7. han 5 minutes after adding solution B1 RNA contamination RNase A not added Add RNase A to buffer A1 to solution Al Plasmid DNA floats Ethanol traces not Make sure that no ethanol residual out of wells while completely removed remaining in the silicon membrane running in agarose from column before eluting the plasmid DNA gel DNA doesn t Re centrifuge or vacuum again if freeze or smell of necessary ethanol Page 10 Express Plasmid Midiprep Optimal Cell Mass OD oo _x mL of Culture This procedure is designed for isolating plasmid grown in standard LB medium Luria Bertani for 12 16 hours to a density of OD o9 2 0 to 3 0 If rich mediums such as TB or 2xYT are used make sure the cell density doesn t exceed 3 0 ODgo0 A high ratio of biomass over lysis buffers result in low DNA yield and purity The column has an optimal biomass of 150 250 For example if the OD oo is 3 0 the optimal culture volume should be 50 to 80 mL Culture Volume Use a flask or tube 4 times bigger in volume than the culture medium to secure optimal condition for bacteria growth Don t exceed the maximum culture volume suggested in the protocol Incomplete lysis due to over amount of bacterial culture results in lower yield and less purity Storage and Stability Buffer A1 should be stored at 4 C once RNase A is added All other materials can be stored at room temperature 22 25 C The Guaranteed shelf life is 12 months
8. nsert to a 2 0 mL tube Spin the column to dry and elute the DNA r e E Express Plasmid Midiprep Page 9 Note It s highly recommended to remove the endotoxin if the DNA is used for endotoxin sensitive cell lines primary cultured cells or microinjection Please use Biomiga s Endofree Plasmid Midiprep Kit PD1420 and PD1422 for transfecting endotoxin sensitive cell lines primary cultured cells or microinjection Limited Use and Warranty This product is warranted to perform as described in its labeling and in Biomiga s literature when used in accordance with instructions No other warranties of any kind express or implied including without limitation implied warranties of merchantability or fitness for a particular purpose are provided by Biomiga Biomiga s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of Biomiga to replace the products Biomiga shall have no liability for any direct indirect consequential or incidental damage arising out of the use the results of use or the inability to use it product Page 8 Express Plasmid Midiprep Kit Contents Catalog PD1468 00 PD1468 01 PD1468 02 Preps 2 10 25 ezBind Columns 2 10 25 Filter Syringe 2 10 25 2 0 ml Microfuge tube 4 20 25 Plastic wrench 1 1 1 Buffer Al 11 mL 55 mL 135 mL Buffer B1 11 mL 55 mL 135 mL Buffer C1 14mL 70 mL 170 mL DNA Washing Buffer 5 mL 24 mL 54mL RNase A 20 mg mL es n o oO an T
9. re and mix the lysate well after adding Buffer C1 Alternatively transfer the lysate to another syringe filter Additional syringe filter can be purchased from Biomiga separately Add 6 mL absolute ethanol 96 100 to the cleared lysate Mix well by sharp shaking for 5 times Add the lysate ethanol mixture into a DNA column set in a 50 mL conical tube Use the plunger to expel the lysate through the column Gently pull the plunger out add 10 mL DNA Wash Buffer expel the Buffer out with the plunger Expel the liquid completely by push and pull of the plunger several times Use the plastic wrench Provided to detach the end component from the midiprep column and insert it into a 2 0 ml eppendorf tube Spin the column at 13 000 15 000 rpm Max speed for 1 min Decant the flow through and put the column back to the tube Spin the column at max speed for 2 min Add 500 uL Elution Buffer Prewarm the Elution Buffer 60 C increases the yield to the center of the column and incubate for 1 min at room temperature Elute the DNA by centrifugation at 12 000 rpm for 1 min Then add 300 uL Elution Buffer to the center of the column and incubate for 1 min at room temperature Elute the DNA by centrifugation at 12 000 rpm for 1 min Repeat step 12 to improve the recovery Optional Add the eluted DNA back to the column for another elution The first elution normally yields about 60 70 of the DNA and the second elution
10. yields another 20 30 of the DNA Note The DNA is ready for downstream applications such as cloning or transfection of HEK293 cells Express Plasmid Midiprep Page 7
Download Pdf Manuals
Related Search