Manual - Xcelris Genomics
Contents
1. Troubleshooting Guide Problems Low Yield Low Yield Low Yield Genomic DNA contamination RNA contamination Plasmid DNA floats out of wells while running in agarose gel DNA doesnt freeze or smellofethanol No phase partitioning after centrigugation Possible Reasons Poor Cell lysis Bacterial culture overgrown or not fresh Low copy number plasmid Over time incubation after adding Buffer B1 RNase A not added to Buffer A1 Ethanol traces were not completely removed from column Temperature is lower than VC Suggestions Resuspend pellet thoroughly by votexing and pipetting prior to adding Buffer B1 Make fresh Buffer B1if the cap had not been closed tightly Buffer B1 0 2M NaOH and 1 SDS Grow bacterial culture upto 12 16 hours Spin down cultures and store the pellet at 20 C if the culture is not purified the same day Do not store culture at 4 C over night Increase culture volume and the volume of Buffer A1 B1 N1 as instructed on page 8 No DNA Plasmid lost in Host E coli Prepare fresh culture Do not vortex or mix aggressively after adding Buffer B1 Do not incubate more than 5 minutes after adding Buffer Add RNase A to Buffer A1 Make sure that no ethanol residue remains in the silicon membrane before elute the plasmid DNA Re centrifuge or vacuum again if necessary Make sure the temperature is greater than 23 C for centrifugation or incubate the sample at 62. 0 C for 5 min and then perform centrifugation Related Products 1 EndoFree plasmid mini kit XG1212 01 2 96Well Plasmid Isolation Kit XG1201 96 3 DNAGel PCR Purification Miniprep kit XG3511 01 XG3514 4 PremixTagV2 0 XG334A 5 Agarose XGA 100 Limited Use andWarranty This product is intended for in vitro research use only Not for use in human This product is warranted to perform as described in its labeling and in XcelGens 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 XcelGen XcelGen s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of XcelGen to replace the products XcelGen 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 For technology support or learn more product information please visit our website at www xcelrisgenomics com XcelGen Quality Kits made by Xperts Plasmid DNA Isolation Kits Genomic DNA Extraction Kits RNA Extraction Kits Polymerase DNA Ladders DNA Markers Premix Tag dNTP s RAPD kits Agarose Glycerol Tms NA Stabilizers 8 RNA Protectant solutions PrimeX Oligo Synthesis amp Purification Services 10 nmole 25 n
3. Buffer A1 Add RNase A to Buffer A1 before use and completely resuspend bacterial pellet by vortexing or pipetting Note Complete resuspension is critical for bacterial lysis and lysate neutralization Add 250ul Buffer B1 mix gently by inverting the tube 10 times do not vortex and incubate at room temperature for 5 minutes Note Do notincubate for more than 5 minutes Note Buffer B1 precipitates cloudy look below room temperature Warm up Buffer B1 at 50 C to dissolve precipitation before use Add 350ul Buffer N1 mix completely by inverting shaking the vial for 5 times and sharp hand shaking for 2 times Note Incubating the lysate in ice for 1 min willimprove the yield Note It is critical to mix the solution well If the mixture still appears conglobated brownish or viscous more mixing is required to completely neutralize the solution Centrifuge the lysate at 13 000 rpm for 10 minutes at room temperature Note If the lysate doesn t appear clean reverse the tube angle centrifuge for 5 more minutes and then transfer the clear lysate to DNA column Ms Add 400ul of Buffer BL into the spin column provided incubate at room temperature for 2 minutes centrifuge at 12000 rpm for 2 minutes and discard the flow through The column is ready and will work well for binding DNA Carefully transfer the clear lysate into a DNA column with a collection tube avoid the precipitations spin at 13 000 rpm for 1 minute dis
4. L z enomics Table of Contents ROCCO mr 02 Sin AC SAIC LAD ARC MPA A 02 UOC NOES coe AN A NEM AO ud EN 02 COMETS coe ey he SM OE coer 04 EVO OEM xc gh FOR 0 MB 04 SEEN ICH ENOL ghd A ec rs U 04 Pes eel Mimi Kie Soin Protocol CR cscs oe one 05 PSN Siotn Macuum Prolocol 4 S 07 Purification of Low Copy Number Plasmid Cosmid sssssssssesseeeseeesees 07 Bunineatiomot plasmides 2b OA A 07 Moubleshooting Glide a Un 09 ake PICIA M A A AU 10 Bimited sc and Wartant A A A U 10 01 xcelri Xce Gen Plasmid Mini Kit ceils An Abellon coo Introduction Key to the kit is our proprietary DNA binding systems that allow the high efficient reversible binding of DNA to the mini column while proteins and other impurities are removed by wash buffer Nucleic acids are then eluted with sterile water or elution buffer This kit is designed for fast and efficient purification of plasmid DNA from 1 to 4ml of E coli culture The mini column has a plasmid DNA binding capacity of 50g The yield from 1 ml cultureis typically around 8to 12pg The purified DNA is ready for downstream applications such as cloning subcloning RFLP sequencing and transfection of robust cells such as HEK293 cells 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 All kit components are stable up to 12 months Important Notes Plasmid Copy Num
5. bers 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 plasmids and expected yield Copy Numbers Expected Yield ug ml Plasmid Origin PSC101 PSC101 0 1 0 2 XcelGen Plasmid Mini Kit xcelris An Abellon company 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 carbohydrates released during lysis We recommend transform plasmid to an endA strain if the yield is not satisfactory Please reference Table 2 for the endA information Table 2 endA strains of E Coli EndA Strains of E Coli EndA Strains of E Coli H M TG1 TB1 ABLE K DH125 LE392 PR700 BL21 DE3 pLysS Cor omes reer size essor mor 0358 All NM strains All Y strains Optimal Cell Mass OD x ml of Culture This procedure is designed for isolating plasmid grown in standard LB medium Luria Bertani for 12 16 hours
6. card the flow through and put the column back to the collection tube Add 500ul Buffer KB into the spin column centrifuge at 13 000 rpm for 1 minute Remove the spin column from the tube and discard the flow through Put the column back to the collection tube Note This step is important to remove residual protein contaminations especially for endA strainsandbehighly ecommended for high guality plasmid DNA Add 650pl DNA Wash Buffer Add ethanol to DNA wash buffer before use into the spin column centrifuge at 13 000 rpm for 1 minute at room temperature Remove the spin column from the tube and discard the flow through Repeat step 10 to improve the recovery Reinsert the spin column with the lid open into the collection tube and centrifuge for 2 minutes at 13 000 rpm Note Residual ethanol can be removed more efficiently with the column lid open It is critical to remove residual ethanol completely Carefully transfer the spin column into a sterile 1 5ml microfuge tube and add 50 100yl sterile ddH O or Elution buffer into the center of the column and let it stand for 2 minutes Elute the DNA by centrifugation at 13 000 rpm for 1 minute Reload the eluate into the column and elute again Note It is recommended to use elution buffer instead of ddH O Note If ddH O is applied please make sure the pH is no less than 7 0 7 0 8 5 is preferred NaOH could be used to adjust the pHof ddH O Note The DNA is ready for dow
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8. e Buffer at 50 C to dissolve the precipitates before use Keep the cap tightly closed for Buffer B1 and Buffer BL after use e Carry outall centrifugation at room temperature Safety Information e Buffer N1 contains acidic acid wear gloves and protective eyewear when handling Buffer N1 and KB contains chaotropic salts which may form reactive compounds when combines with bleach Do not add bleach or acidic solutions directly to the preparation waste 04 ELE Plasmid Mini Kit Spin Protocol ile 05 Inoculate 1 4ml LB containing appropriate antibiotic with a fresh colony from a freshly streaked selective plate Incubate at 37 C for 14 16 hours with vigorous shaking Note Prolonged incubation gt 16 hours is not recommended since the E coli starts to lyse and the plasmid yields may be reduced Note Do notgrow the culture directly from the glycerol stock Note This protocol is optimized for E coli strain cultured in LB medium When using TB or 2xYT medium special care needs to be taken to ensure the cell density doesn t exceed 3 0 OD Buffers need to be scaled up proportionally if over amount of cultures are being processed Harvest the bacterial culture by centrifugation for 1 min at 10 000 rpm Pour off the supernatant and blot the inverted tube on a paper towel to remove residue medium Remove the residue medium completely Note Residue medium will cause Poor cell lysis and thus lower DNA yield Add 250ul
9. mole 50 nmole 100 nmole 200 nmole 1000 nmole NGS Services Denovo Genome Sequencing Whole Genome Resequencing GBS RAD Sequencing Exome Sequencing Amplicon Sequencing Whole Transcriptome Analysis RNA Sequencing Small RNA Sequencing Metagenomics Metatranscriptomics ChIP Sequencing Mitochondrial Sequencing Next Generation Genomic Services on Illumina MiSeq Genotyping by Sequencing Tilling Ecotilling using NGS Genome Database development Services NGS Bioinformatics e In silico Primer Design Microarray Analysis Metagenomics Physical Genetic and QTL mapping Assembly and annotation of prokaryotic and eukaryotic genome Genome Mapping and SNP discovery Transcriptome discovery and analysis sRNA analysis and discovery XcelSeq Sanger Sequencing Servi Plasmid PCR Sequencing Services r E coli Culture Sequencing Services Primer Walk Sequencing Services Microbial Identification Service Multilocus Sequence Typing Customised Services SNP Genotyping by SNaPshot Assay Microsatellite Genotyping Golden Gate Assays and Arrays Gene Expression on Real Time PCR Gene expression on Agilent Microarray Affymetix Library construction Xcelris Labs Limited e xce rl S Old Premchand Nagar Road Opp Satyagrah Chhavani Bodakdev Ahmedabad 380015 India Tel 91 79 66197777 Fax 91 79 66309341 genomics Website www xcelrisgenomics com An Abellon company E mail bdgenomics x
10. nd for 2 minutes Elute the DNA by centrifugation at 13 000 rpm for 1 minute Reload the eluate into the column and elute again Note It is recommended to use Elution Buffer instead of ddH 0 Note The DNA is ready for downstream applications such as cloning RFLP library screening in vitro translation sequencing and transfection of robust cells such as HEK293 cells Fig Agarose gel analysis of plasmid DNA purified with XcelGen Plasmid mini Kit Lane 1 pbluescript Il Lane 2 pUC 18 Lane 3 pBR 322 Lane 4 pGEM Lane 5 pET 43 1 LaneL 1 kbladder Purification of Low Copy Number Plasmid 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 Culture volume Use 2 x volumes of the high copy number culture Use 2 x volumes of the Buffer A1 Buffer B1 and Buffer N1 Additional Buffers can be purchased from XcelGen Use same volume of DNA Wash Buffer and Elution Buffer Purification of plasmid gt 12kb For isolating plasmid DNA gt 12 kb use the following guideline Culture volume Use 2 x volumes of the culture Use 2 x volumes of the Buffer A1 Buffer B1 and Buffer N1 Additional Buffers can be purchased from XcelGen Use same volume of DNA Wash Buffer and Elution Buffer Pre warm the Elution Buffer at 65 70 C and let the column stand for 5 minutes after adding Elution Buffer 08
11. nstream applications such as cloning subcloning RFLP library screening in vitro transfection sequencing transfection of robust cells suchas HEK293 cells Note It is highly recommended to remove the endotoxin XG1212 01 if the DNA is used for endotoxin sensitive cell lines primary cultured cells or microinjection The DNA concentration can be calculated as follows Concentration ug ml OD nm X 50 X dilution factor 06 Plasmid Mini Kit Spin Vacuum Protocol 1 07 Set up the vacuum manifold according to manufacture s instruction and connect the column to the manifold Carry out step 1 7 on Page 6in previous protocol Carefully transfer the clear lysate to the DNA column and turn on the vacuum to allow the lysate pass through the column Add 500ul Buffer KB into the spin column and allow the lysate pass through the column by vacuum Note This step is important to remove residual protein contaminations especially for endA strains and be recommended for high quality plasmid DNA Add 650yl of DNA Wash Buffer to the column and allow the vacuum to draw the liquid through the manifold Turn o the vacuum Repeat step 5 to improve the recovery Transfer the column with the lid open to a 1 5 ml collection tube and centrifuge at 13 000 rpm for 2 minutes Carefully transfer the spin column into a clean 1 5 ml microfuge tube and add 50 100pl gt 50ul Sterile ddH 0 or Elution Buffer into the column and let it sta
12. to a density of OD 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 OD A high ratio of biomass over lysis buffers result in low DNA yield and purity The mini column has an optimal biomass of 10 15 For example if the OD is 3 0 the optimal culture volume should be 1 5 ml For over amount of cell numbers either reduce the biomass or scale up the volumes of Buffer A1 B1 and N1 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 xcelri Xce Gen Plasmid Mini Kit ceils An Abellon company Kit Contents Product XG1211 00 XG1211 01 DTA Before Starting Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each steps and pay special attention to the followings Important RNase A 20mg ml 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 A1 and mix well before use Store at 4 C Add 8ml XG1211 00 or 60 ml XG1211 01 or 96 100 ethanol to each DNA Wash Buffer bottle before use Buffer B1 and Buffer BL precipitates below room temperature It is critical to warm up th
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