Kit Manual
Contents
1. Plasmid Origin Copy Numbers Expected Yield ug per 500 mL pSC101 pSC101 5 50 60 pACYC P15A 10 12 80 100 pSuperCos pMB1 10 20 80 150 pBR322 pMB1 15 20 100 150 pGEM Muted pMB1 300 400 2000 2500 pBluescript ColE1 300 500 2000 3000 pUC Muted pMB1 500 700 3000 4000 Page 2 Biomiga EZgene Plasmid Mega 10 Kit Host Strains The strains used for propagating plasmid have significant influence on yield Host strains such as Top 10 and DHSa 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 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 PD1714 Table2 endA strains of E Coli DH5a DHI DH21 JM106 JM109 SK2267 SRB XLO TOPIO DHIOB 3M103 JM107 SK1590 MM204 s m Xi BJ5182 DH20 JM105 M108 SK1592 Selectog stpm XET C600 CJ236 KW251 P2392 BL21 DE3 C HB101 TG1 TB1 ABLE pale LE392 PR700 BL21 DE3 K TM pLyssS JM101 JM83 TKB1 HMS174 ES1301 M1061 Q358 BMH 71 18 All NM strains All Y strains Optimal Cell Mass OD o _x_ mL of Culture This procedure is designed for isolating plasmid grown in standard LB medium Luria Bertani for 122. Biomiga EZgene Plasmid Mega 10 Kit Genomic DNA contamination Over time incubation after adding buffer Bl Do not vortex or mix aggressively after adding Buffer B1 Do not incubate more than 5 minutes after adding solution B1 RNA contamination RNase A not added to Buffer Al Add RNase A to Buffer Al Plasmid DNA floats out of wells while running in agarose gel DNA doesn t freeze or smell of ethanol Ethanol traces not completely removed from column Make sure that no ethanol residual remaining in the silicon membrane before eluting the plasmid DNA Re centrifuge or vacuum again if necessary Biomiga EZgene Plasmid Mega 10 Kit Page 11
3. 16 hours to a density of OD 09 2 0 to 3 0 If rich medium such as TB or 2xYT are used make sure the cell density doesn t exceed 3 0 ODg 09 A high ratio of biomass over lysis buffers result in low DNA yield and purity Culture Volume Use a flask or tube with a volume at 4 times 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 Biomiga EZgene Plasmid Mega 10 Kit Page 3 Table 3 The optimal cell mass culture Volume and Binding Capacity for the mega DNA units DNA Units Mega 3 Mega 6 Mega 10 Optimal Cell Mass 1200 2500 4500 Culture Volume 500 mL 1000 mL 1500 mL Binding Capacity 3 4 mg 6 7 mg 10 12 mg Storage and Stability Buffer Al 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 from the date of purchase Before Starting Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each steps Important e RNase A It is stable for more than half a year when stored at room temperature Spin down RNase A vial briefly Add the RNase A solution to Buffer Al and mix well before use e Buffer B1 precipitates below room temperature It
4. Contents Contents Introduction Important Notes Storage and Stability Before Starting Important Materials supplied by users Kit Contents Safety Information EZgene Plasmid ezFilter Megaprep 10 Protocol Purification of Low Copy Number Plasmid and Cosmid Trouble Shooting Guide a AR NN NK A uw 6 amp 6 A A U 10 Biomiga EZgene Plasmid Mega 10 Kit Page 1 Introduction Key to the kit is our proprietary DNA binding systems that allow the high efficient binding of DNA to our ezBind matrix while proteins and other contaminates are removed under certain optimal conditions Nucleic acids are easily eluted with sterile water or elution buffer Unlike other procedures our patented plasmid purification kit has no guanidine salt in the buffer the purified DNA is guanidine ion exchange resin residues free which enable the high performance of downstream applications such as transfection 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 to 3 times Reference Table 1 for the commonly used plasmids Table 1 Commonly used plasmids
5. ace the 500 mL or 1 000 mL Page 8 Biomiga EZgene Plasmid Mega 10 Kit bottle with a sterile 50 mL conical tube screw tight 14 Add 12 mL sterile ddH O or Elution Buffer evenly to the membrane and incubate for 2 minutes Turn on vacuum to elute DNA Typically 5 6 mL of DNA containing solution can be collected This is the 1 elution 15 Turn off the vacuum and replace the 50 mL conical tube with another sterile 50 mL conical tube screw tight Add 12 mL sterile ddH O or Elution Buffer and incubate for 1 minute Turn on the vacuum and collect the 2 elution typically 8 10 mL of solution can be collected Note If ddH O is used for eluting DNA make sure the pH is 7 0 Note The DNA is ready for downstream applications such as cloning subcloning RFLP Library screening in vitro translation sequencing transfection of robust cell lines HEK293 cells Note It s highly recommended to remove the endotoxin PD1615 if the DNA is used for endotoxin sensitive cell lines primary cultured cells or microinjection Note Two elutions give rise to maximum DNA yield For maximum yield and higher concentration pool the elutions together add 0 1 volume 3M KAc or NaAc pH 5 2 and 0 7 volume isopropanol Centrifuge at top speed for 10 min Discard supernatant Wash the DNA with 1000 uL 70 ethanol centrifuge for 5 min carefully decant Air dry the pellet for 10 20 minutes in a tissue culture hood Resuspend the DNA in Elution Buff
6. er or sterile ddH O DNA concentration ug mL OD2 0 nm X 50 x dilution factor Purification of Low Copy Number Plasmid and Cosmid The yield of low copy number plasmid is normally around 0 1 1 ug mL of overnight culture For isolating low copy number or medium copy number plasmid DNA use the following guideline Biomiga EZgene Plasmid Mega 10 Kit Page 9 1 Culture volume Use 2 x volumes of the high copy number culture 2 Use 2 x volumes of the Buffer A1 Buffer B1 and Buffer C1 and 100 ethanol Additional buffers can be purchased from Biomiga 3 Use same volume of Wash Buffer 70 ethanol and 100 ethanol and Elution Buffer Trouble Shooting Guide Problems Possible Reasons Suggested Improvements Low Yield Poor Cell lysis e Resuspend pellet thoroughly by votexing and pipeting prior adding Buffer B1 e Make fresh buffer B1 if the cap had not been closed tightly Buffer B1 0 2N 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 40C over night Low Yield Low copy number Increase culture volume to 2 x of plasmid original volume Increase the volume of buffer Al B1 C1 and ethanol proportionally with the ratio of 1 1 1 2 1 2 No DNA Plasmid lost in Host Prepare fresh culture E coli Page 10
7. ing for 10 times till a flocculent white precipitate forms Incubate the mixture at room temperature for 10 minutes Note It is critical to mix the lysate well If the mixture still appears conglobated brownish or viscous more mix is required to completely neutralize the solution Attach the 2 layer filter unit to a sterile 500 mL or 1000 mL standard bottle Corning 430518 or 430282 or equivalent pyrex glass bottle and screw tight Connect the unit to a pump driven vacuum system Transfer the clear lysate from the bottom of the mixture use a 50 mL serological pipet to the filter unit Stand by for 5 minutes and turn on the vacuum with low vacuum force and increase to maximum vacuum force after 5 minutes Note 1 Low vacuum force prevents clogging of the filter membranes Note 2 Use a 50 mL serological pipet to transfer the relatively clear lysate from the bottom of the lysate bottle to the filter unit This will speed up the flow rate of the filter unit Normally around 80 mL lysate can be filtered through the filter unit within 10 15 Filter Assembling Right Wrong W AX Biomiga EZgene Plasmid Mega 10 Kit Page 7 10 11 12 13 minutes Pour the remaining white precipitates to the filter unit when most of the lysate has been filtered through Figure 1 Instruction of filter assembling Note 3 If the flow through gets too slow turn off the vacuum and wait for 1 minute Carefully detach the uppe
8. is critical to warm up the buffer at 50 C to dissolve the precipitates before use e Keep the cap tightly closed for Buffer B1 after use e The proper volume of buffer ratio of A1 B1 C1 100 ethanol 1 1 1 2 1 2 Page 4 Biomiga EZgene Plasmid Mega 10 Kit e Make sure the availability of centrifuge and vacuum manifold especially after mixing the lysate with ethanol the sample needs to be processed immediately by vacuum Materials supplied by users e 70 ethanol and 100 ethanol e Pump driven vacuum system 500 mL bottle or 1 000 mL bottle Corning 430518 or 430282 or equivalent pyrex glass bottles e 50 mL conical tubes Kit Contents Catalog PD1614 00 PD1614 01 PD1614 02 Preps 1 2 10 DNA Unit 1 2 10 Filter Unit 1 2 10 Replacement Cup 1 4 20 Buffer Al 110 mL 210 mL 2 x 530 mL Buffer B1 110 mL 210 mL 2 x 530 mL Buffer C1 130 mL 250 mL 3 x 450 mL RNase A 20 mg mL ean re mL 4 F Elution Buffer 30 mL 60 mL 270 mL User Manual 1 1 1 Biomiga EZgene Plasmid Mega 10 Kit Page 5 Safety Information e Buffer Cl contains acidic acid wear gloves and protective eyewear when handling e 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 EZgene Plasmid ezFilter Megaprep 10 Protocol 1 Inoculate 1 200 1 500 mL LB containing ap
9. propriate antibiotic with 500 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 The buffer volumes need to be scaled up if processing over 2 000 mL of culture Note Do not use a starter culture that has been stored at 4 C Note Do not grow starter culture directly from glycerol stock 2 Harvest 1 200 1 500 mL overnight bacterial cells by centrifugation at 5 000 x g for 10 minutes at room temperature Decant or aspirate medium and discard Note Remove the residual medium completely for optimal cell lysis and neutralization 3 Resuspend the bacterial pellet in 100 mL Buffer A1 Add RNase A to Buffer Al before use Pipet or vortex till the bacterial pellet dispersed thoroughly Complete resuspension is critical for optimal yields 4 Add 100 mL Buffer B1 mix gently but thoroughly by inverting 10 times and incubate at room temperature for 5 minutes to obtain a cleared lysate Note Do not incubate longer than 5 minutes Over incubating causes genomic DNA Page 6 Biomiga EZgene Plasmid Mega 10 Kit contamination and plasmid damage Avoid vigorous mixing as this will shear the genomic DNA Add 120 mL Buffer C1 and mix immediately by inverting 5 times and sharp hand shak
10. r filter cup and replace it with the replacement cup Assemble the unit as Figure 1 Pour the lysate from the original cup to the replacement cup Turn on the vacuum and filter the rest of the lysate When most of the lysate has been filtered through the unit turn off the vacuum wait for 1 minute detach the unit and discard the upper filter cup including the rubber rings Note The DNA is in the collection bottle Connect the DNA unit to a 500 mL or 1 000 mL standard bottle and screw tight Connect the DNA unit to the vacuum with the vacuum off Add 120 mL 100 ethanol to the lysate bottle Mix well by sharp hand shaking 3 5 times and immediately pour half of the lysate ethanol mixture to the DNA unit and turn on the vacuum Pour the rest of the lysate ethanol mixture into the DNA unit When all the lysate pass through the DNA unit vacuum for 1 minute Wash the DNA membrane with 50 mL 70 ethanol and vacuum for minute at maximum force Repeat this step once Add 80 mL 100 ethanol evenly to the DNA membrane and vacuum for 1 minute Turn off the vacuum wait for 1 minute and discard the liquid waste in the bottle Reconnect the bottle to the DNA binding unit Turn on the vacuum for 20 minutes at maximum force to remove the ethanol residues Note Residual ethanol can be removed more efficiently with the column lid open It is critical to remove residual ethanol completely Turn off the vacuum wait for 1 minute and repl
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