Minicircle DNA Vector Technology Cat. #MNXXX Series
Contents
1. C31 integrase and the I Scel endonuclease simultaneously The C31 integrase produces the MC DNA molecules and PP DNA from the full size MC DNA upon arabinose induction The PP DNA contains a number of engineered I Scel restriction sites that are subject to the digestion of I Scel endonuclease and ultimate destruction of the PP DNA The MC DNA is lacking I Scel restriction site so that it remains intact The 32 copies of l Scel sites in the PP DNA secure the production of superclean MC DNA without PP DNA contamination This bacterial strain produces purified MC DNA in a time frame and quantity similar to those of routine plasmid DNA preparation Minicircle DNA vectors allow sustained trangene expression in quiescent cells and tissues These vectors have been demonstrated 10 to 1 000 fold enhancement compared to regular plasmids in long term transgene expression in quiescent tissues in vivo and in vitro The mechanism of enhanced transgene expression may result from eliminating heterochromatin formation induced by the plasmid backbone and methylation and transgene silencing The major obstacle to the widespread use of mincircles has been the time consuming labor intensive production The MC Easy System from SBI enables a simple reproducible and efficient way to produce high quality Minicircle DNA The finely tuned growth and induction media produces minicircle DNA that is free of parental and genomic DNA contamination The Kit also includes an addi2. Mix by vortexing vigorously for a few seconds Incubate at room temperature for 15 min Add the mixture to one well of a 12 well plate and incubate at 37 C 5 CO overnight Check for fluorescent reporter protein expression e g GFP or RFP or other marker gene or protein of interest expression J Tail Vein injection of Minicircles for in vivo expression of transgenes Minicircle DNA can be injected into mice by tail vein injection for in vivo expression of transgenes We recommend following the protocols set up by your institution s animal handling facility 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual Page 14 ver 5 121016 www systembio com Minicircle DNA Technology Cat MNxxxA 1 IV Example Data The gel image below shows uninduced minus arabinose and induced plus arabinose ZYCY10P3S2T E coli The resulting minicircles 3kb are produced Arabinose M 10kb fp kb mc Transfection of 1 ug minicircle DNA pMC CMV MCSOEF1 GFP SV40PolyA into HEK293 cells Expression of GFP persists for at least 8 days 6 Hours 2 Days 4 Days 5 Days 6 Days 8 Days Example data of in vivo transgene expression in a mouse injected with minicircle DNA through tail vein injection Plasmid DNA is degraded after 2 weeks of expression while minicircle DNA expression remains strong for at least 3 weeks o EB Minicircle DNA J Plasmid DNA E 8 Tra
3. 1 On day 1 at about 5 00 PM inoculate 2 ml of LB containing 50 g m kanamycin with a single colony or an inoculation loop of glycerol stock bacteria Incubate at 30 C shaking at 250 rpm for 1 2 hours Note The bacteria must be fresh LB agar kanamycin plates stored at 4 C are only fresh for 1 week Glycerol stocks are good for up to one year 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual 2 After 1 2 hrs measure the ODso to calculate the inoculate volume Use the following formula to calculate inoculate volume 2 x 1074 Inoculate volume ml ODsoo For example if the ODsoo reading is 0 01 the inoculate amount is 2x 1074 I late vol D noculate volume ml 001 Inoculate volume ml 0 02 ml 20 ul Dilute 5X Growth medium to 1X 160 ml of sterile deionized water 40 ml of 5X Growth Medium 200 ml Total volume 1X Growth Medium Inoculate 200 ml of 1X Growth Medium with inoculate volume calculated in step 2 Grow bacteria overnight at 30 C shaking at 250 rpm in a 1 liter sterile flask The overnight culture should not go beyond 16 hrs If using a different sized flask for the overnight culture keep the ratio of flask size to O N culture volume at 5 1 vol vol Page 8 ver 5 121016 www systembio com Minicircle DNA Technology Cat MNxxxA 1 Day 2 Induction 1 On Day 2 after about 16 hrs of overnight growth measure the pH and ODe
4. resuspension buffer lysis buffer and neutralization buffer Consider adding LyseBlue reagent to resuspension buffer Solution should turn evenly blue after adding lysis buffer and Page 18 ver 5 121016 www systembio com Minicircle DNA Technology Cat MNxxxA 1 inverting 4 5 times VI References Kay et al A robust system for production of minicircle DNA vectors Nature Biotechnology 2010 Fangjun Jia et al A nonviral minicircle vector for deriving human iPS cells Nature Methods 2010 Mar 7 3 197 9 Zhi Ying Chen et al Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo Human Gene Therapy 16 1 126 131 January 2005 Zhi Ying Chen et al Minicircle DNA Vectors Devoid of Bacterial DNA Result in Persistent and High Level Transgene Expression in Vivo Molecular Therapy 8 3 495 500 September 2003 888 266 5066 Toll Free 650 968 2200 outside US Page 19 System Biosciences SBI User Manual Vil Vector Maps This is a partial list of available minicircle Parental Plasmids A complete list can be found on SBI s website Full sequences of the parental plasmids can be obtained by contacting SBI Expression cassette options swa suso Catf am MH Mnso1a 1 ETS MN502A 1 ao E MANS 1141 a a MN512A 1 MNS30A 1 E LEE EO MNs31A 1 CMV AGE EN MN601A 1 ME MN602A 1 Catalog De
5. Centrifugal columns 12 Transfect Minicircle DNA with MC Fection to validate the construct 13 J Tail Vein injection of Minicircles for in vivo expression of transgenes 13 IV Example Data ccieicccscsecascccvasddtenecdacgstadedcaatnccadearagegaens 15 V Troubleshooting 3 a isani diia 17 VI EEEE E E E is audios dadadei TE 19 VII Vector Maps pensee iina iana ENE AEEA ARNE 20 VIII Technical Support er 21 IX Licensing and Warranty 21 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual I Introduction and Background A The Minicircle Technology Minicircles MC are circular DNA elements that no longer contain antibiotic resistance markers or the bacterial origin of replication These small vectors can be used in vivo or in vitro and provide for long term transient expression of one or more transgenes without the risk of immunogenic responses that can be caused by the bacterial backbone in standard plasmids Production of minicircles requires a special parental plasmid and an engineered E coli strain that allows both propagation of the parental plasmid and the production of the minicircles Minicircles are conditionally generated by an expression of inducible C31 integrase via intramolecular cis recombination The full size MC DNA construct is grown in a special host E coli bacterial strain that harbors an Arabinose inducible system to express the
6. Page 12 ver 5 121016 www systembio com Minicircle DNA Technology Cat MNxxxA 1 7 Insert the 2ml DNA Centrifugal filter into a filtrate collection tube Add up to 2 ml of sample dissolved in TE to the device and cover with concentrate collection tube Place filter device into the centrifuge rotor and counterbalance with a similar device a For a swinging bucket rotor spin at 1 000x g for 30 min or 3000x g for 10 min b Fora fixed angle rotor spin at 7 500 x g for 10 30 min Wash step add 2 ml of the buffer you want and repeat spin procedure Remove the assembled device from the centrifuge and separate the 2ml DNA Centrifugal column from the filtrate collection tube To recover the concentrated solute invert the filter column to concentrate collection tube Spin for 2 minutes at 300 1 000 x g to transfer the concentrated sample from the column to the tube NOTE For optimal recovery perform the reverse spin immediately Measure plasmid concentration and check A260 A280 ratio The A260 A280 ratio should be 1 8 1 9 Transfect Minicircle DNA with MC Fection to validate the construct 1 Seed 1 5X10 target cells ml per well in a 12 well plate SBI uses 293T cells but you can use any target cells that you prefer 2 Incubate overnight at 37 C Cells should be 50 70 confluent the next day 3 In a sterile Eppendorf tube add the following per well 100 ul DMEM without FBS 1 ug Minicircle DNA 2 5 yl MC Fection
7. for minicircle production as they do not have the appropriate genome modifications to produce minicircles C Minicircle shRNA Expression Vectors The Minicircle shRNA parental plasmids contain either a CMV or EF1 promoter that drive expression of GFP or GFP and Puromycin resistance followed by an H1 promoter that drives expression of the shRNA construct E Reverse yz Sune Loop Antisense Terminator tecGACTCCAGTGGTAATC TACcttcctgtcagsGTAGATTACCACTGGAGTCtttttgaa aggCTGAGGTCAC CATTAGATGgsaggecagt ct CATCTAATGGTGACC TCAGasaaactt Transcription tt S GACUCCAGUGGUAAUCUAC t shRNA transcript g 3 uuCUGAGGUCACCAUUAGAUG Sac Typically 3 5 target sequences in the gene of interest need to be selected and tested to identify functional shARNAs with at least 70 silencing efficiency of the target mRNA Although there is no standard rule for selecting the target mRNA binding sites for shRNA sequences we have found the following criteria useful e 19 29 nt in length usually longer oligos 25 27 nt are more robust and give better silencing efficiencies although 19 nt oligos could be also used e Unique sequences with less than 70 homology with other mRNA sequences in a RefSeq database Especially avoid homology to other non target mRNA sequences in the central portion of shRNA Flanking sequences usually tolerate mismatches without reduction in silencing efficiency e 40 55 GC content 888 266 5066 Toll Free 650 968 220
8. 0 outside US Page 3 System Biosciences SBI User Manual No more than 4 consecutive A s or T s No more than 5 consecutive G s or C s No thermodynamically stable secondary structure 0 Kcal mol A 5 terminus 3 5 flanking nucleotides on the antisense strand should be more AT rich than the 3 terminus The template sequences coding for the shRNA targeted to each selected target site must contain both the sense and anti sense strand and be designed to form a stem loop structure when transcribed In addition both the top and bottom strands of the entire shRNA sequence sense loop antisense terminator must be synthesized and annealed to make a double stranded DNA sequence that can be cloned into the vector The features of the oligonucleotides coding for the shRNA template sequence should include the following The 19 29 nucleotide sense and antisense mRNA sequences Usually longer siRNAs 25 27 nt have better silencing efficiencies although 19 nt oligos are more commonly used A hairpin loop sequence between sense and antisense portion The 9 nt loop sequence 5 TTCAAGAGA 3 is most commonly used in RNA silencing experiments but we have used a 12 nt sequence 5 CTTCCTGTCAGA 3 which generates similar results Loop sequences of 3 to 15 nucleotides have been used successfully by different investigators A TTTTT terminator sequence for RNA polymerase III A BamHI and EcoRI restriction site overhang sequences for directi
9. NIN ll Non intggrative sustained expression JAS System Biosciences Minicircle DNA Vector Technology Cat FMNXXX Series User Manual Check Individual PACs for storage conditions A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement ver 5 contained in this user manual Minicircle DNA Technology Cat MNxxxA 1 Contents I Introduction and Background 2 A The Minicircle Technology cocino 2 B ZYCY10P3S2T E coli eene eene neee reer reer nerr eee 3 C Minicircle shRNA Expression Vectors 3 Il Minicircle Cloning Protocols cccccceeseeeeeeeeneeeeeeeneeeeteeneees 4 Cloning into Minicircle Parental Plasmids 4 B Cloning into Minicircle shRNA Parental Plasmids 4 III Minicircle Production Protocol 6 A Contents rear nrcnnnannncnnnnns 6 B Transforming ZYCY10P3S2T E coli Minicircle producer strain MN900A 1 7 C ROCOVETY ssc eeeeeeceeeeeeeeeeeneeeeeeeeaeeeeeeaeeeeeeaeeeeeeaaeeeeeenaneeeeeaas 7 D Plating and Mini Preps 7 E Growth and Induction of the Minicircle Producer Strain 7 F Removal of Genomic and Parental Plasmid DNA contamination 12 G Removal of inactivated DNase and Restriction Enzymes 12 H Optional Removal of ATP and dNTPs with 2ml DNA
10. ction of Minicircle plasmid Follow protocol of Invitrogen s PureLink HiPure Plasmid Purification Kit but use 2 3 Maxi columns for each minicircle construct produced Digest 0 5 ug minicircle plasmid with a restriction enzyme and run an agarose gel to check the quality again If the quality is good go to the transfection procedure offered in Section to check functionality of the construct If the minicircle preparation contains genomic DNA or parental DNA or both continue with the next steps Sections F H of this protocol a If the parental DNA contamination is more than 10 higher than the minicircle DNA yield you must start again Please refer to the troubleshooting section for more details 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual b It is okay to proceed with excess genomic DNA contamination in the absence of parental DNA contamination This is an example of a purified minicircle plasmid that does not have any genomic or parental plasmid contamination If your preparation looks like this you can proceed by transfecting the minicircles into your cells of interest Fig 1 Example of minicircle prep w o genomic or parental DNA contamination Lanes 2 4 DNA Ladder L1 L2 13 L4 L1 Parental DNA of SRM100PA 1 cut by Mfe1 L2 L3 L4 Minicircle DNA of SRM100PA 1 cut by Mfe1 Page 10 ver 5 121016 www systembio com Minicircle DNA Technology Cat MNxxxA 1 Th
11. es arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty SBI does not 888 266 5066 Toll Free 650 968 2200 outside US Page 21 System Biosciences SBI User Manual provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2012 System Biosciences SBI All Rights Reserved Page 22 ver 5 121016 www systembio com
12. fer 20 ul DNase 10 pl water 500 ul Total Reaction Volume Incubate at 37 C for 2 16 hours 3 Inactivate DNase by incubation at 70 C for 30 minutes 4 Check Result Take 1 2 ul from DNase treated and untreated samples Digest them with same restriction enzyme and run agarose gel to check the result If the reaction was successful you should see minicircle DNA present but no parental plasmid or genomic DNA contamination in the sample G Removal of inactivated DNase and Restriction Enzymes Add 50 ul of Precipitation Buffer 1 10 the volume of the reaction in the previous step to the DNase treated solution and mix by pipeting Add 1 1 ml of 100 ethanol and mix by inverting Put on ice for 30 min Centrifuge at 15000x g for 15 minutes Carefully discard supernatant Do not disturb DNA pellet Dissolve plasmid with 1 2 ml of TE pH 7 5 buffer if using centrifugal columns see below Alternatively if presence of ATP and dNTPs do not influence your downstream experiment you can wash pellet with 1 ml of 70 ethanol Centrifuge at 15000x g for 5 minutes Discard supernatant When pellet is dry resuspend in endotoxin free water supplied or endotoxin free TE pH 7 5 to 8 0 buffer H Optional Removal of ATP and dNTPs with 2ml DNA Centrifugal columns 2ml DNA Centrifugal columns can be used to remove dNTPs primers salts and buffers and other macromolecular components Ho Uo Fill Spin Recover Collect Tr
13. is is an example of a minicircle plasmid that has both genomic DNA and parental plasmid contamination If your minicircle preparation looks like this you will need to perform the Removal of Genomic and Parental Plasmid DNA contamination steps Section F before transfecting it into your cells of interest Fig 2 Example of minicircle prep showing genomic parental DNA contamination Lane 1 and after treatment with minicircle safe DNase Lane 2 DNA Ladd L1 L2 Parental DNA Minicircle F L1 Minicircle DNA contaminated with genomic DNA and parental DNA L2 Minicircle DNA treated with Nde1 and Minicircle safe DNAse 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual F Removal of Genomic and Parental Plasmid DNA contamination Parental DNA and genomic DNA contamination are common problems in minicircle DNA production Follow this protocol to remove parental plasmid and genomic DNA contamination 1 Linearize parental DNA with 1 or 2 restriction enzymes that cut the bacterial backbone without cutting inside the minicircle DNA You should use a plasmid editor software to help you choose the appropriate restriction enzymes 2 Then use Minicircle safe DNase to digest the linearized DNA according to the protocol below Minicircle safe DNase is included in the kit 400 ul plasmid use the whole amount you obtained in the last step 20 pl 25 mM ATP 50 ul 10X Reaction Buf
14. mpletely 888 266 5066 Toll Free 650 968 2200 outside US Page 17 System Biosciences SBI User Manual Solution was not mixed soon enough after the addition of buffer P3 This could result in the formation of clusters of PDS potassium dodecyl sulfate together with cellular debris including genomic DNA These clusters have a tendency to float If these floating pieces are added toa column then genomic DNA contamination almost definitely will occur Mix immediately after adding Buffer P3 or N3 or S3 by inverting vigorously 4 6 times Remove floating pieces by filter Do not overload the column beyond its Capacity Parental DNA contamination Bacteria is stored at 4 C for more than one week pH lt 6 5 before induction Always use fresh bacteria Check pH and correct to gt 6 5 before induction Cold induction medium or temperature during induction is too low Use room temperature induction medium Induction time is not enough or too long Adjust induction time gt 10 parental plasmid contamination Start the protocol from the beginning Low yield The plasmid failed to precipitate which is especially common when the size of minicircle DNA lt 3 5 kb Use high speed centrifuge 15000G for 30 60min to precipitate small size minicircle DNA Bacteria was not resuspended or lysed completely Use double amount of
15. nsgene Expression Level 48 Hours 2 Weeks 3 Weeks 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual Page 16 ver 5 121016 www systembio com Minicircle DNA Technology V Troubleshooting The most common problems in minicircle production are 1 Genomic DNA contamination 2 Parental DNA contamination Cat MNxxxA 1 3 Low yield Problems Possible causes Solutions Genomic DNA Bacteria over growth Shorten culture time and contamination reduce the amount of innoculum Check ventilation and shake speed Bacteria is not fresh or was stored at 4 C for gt 1 week Always use fresh bacteria Ventilation is not good Keep the ratio of flask size to culture volume at 5 1 vol vol Culture medium pH is too low or incubation time is too long Keep the culture pH gt 6 5 Mechanical damage to bacteria such as vigorously vortexing after freezing Resuspend bacteria pellet carefully and completely after freezing by gently vortexing and pipetting up and down Lysis time gt 5min Invert bottles gently after adding lysis buffer and keep at RT lt Smin Insufficient quantity of SDS in lysis buffer to permit complete binding to the cellular debris When the buffer is stored at temperatures below 20 C which causes the SDS to precipitate out of solution Warm Lysis buffer at 50 C a few minutes to dissolve the SDS co
16. omic and parental DNA contamination The system comes complete with a special minicircle DNA transfection reagent MC Fection that works well with most cell lines Choose from the minicircle production kit that includes the engineered E coli producer strain ZYCY10P3S2T cat MN920A 1 or select the minicircle production and isolation kit alone MN910A 1 A Contents The MC Easy Minicircle DNA Production kit provides enough reagents for 5 minicircle production preparations 5X Minicircle Growth medium 200 ml 10X Induction Medium 200 ml Endotoxin free water 5 ml Minicircle safe DNase 125 ul ATP 25mM 125 ul 10X DNase buffer 250 ul Precipitation Buffer 0 5 ml 2 ml DNA Centrifugal Filter Columns 5 filters MC Fection transfection reagent for minicircle DNA 100 ul NS 5 vials Other Reagents Needed but not included in the kit e Sterile deionized water e 100 Ethanol e LB agar Kanamycin plates Page 6 ver 5 121016 www systembio com Minicircle DNA Technology Cat MNxxxA 1 B Transforming ZYCY10P3S2T E coli Minicircle producer strain MN900A 1 1 Thaw competent cells on ice 2 Add DNA from ligation reaction using 1 5 ul of the reaction to one vial of ZYCY10P3S2T cells moving the pipette through the cells while dispensing Gently tap tubes to mix 3 Alternatively for parental plasmid transformation add 20 100 ng DNA to the vial 4 Incubate cells on ice for 30 minutes 5 Heat shock cells f
17. onal cloning of annealed shRNA template oligonucleotides into the vector Using of initiation G nucleotide in the first position of sense portion of SHRNA is not necessary as RNA polymerase III could initiate transcription from any 1 nucleotide of the H1 promoter The top and bottom strands of the shRNA template oligonucleotides should be designed to look like the following diagram after annealing RNA Pol Ill BamHi Sense Strand Loop Antisense Strand Terminator EcoRI GATCCNNNNNNNNNNNNNNNNNNNCTTCCTGTCAGANNNNNNNNNNNNNNNNNNNTITTTG 3 3 GNNNNNNNNNNNNNNNNNNNGAAGGACAGTCTNNNNNNNNNNNNNNNNNNNAAAAACTTAA 5 Minicircle Cloning Protocols Cloning into Minicircle Parental Plasmids Clone your insert of interest into the parental plasmid of choice either by using the multiple cloning site or by using SBI s Cold Fusion cloning kit Cat MC010 MC100 or MC101 Sequences of the multiple cloning sites for the different parental plasmid vectors can be obtained from SBI Your insert should contain a Kozak sequence ATG start site and stop codon Propagation of the parental plasmid during cloning and screening can be accomplished with any strain of competent E coli or you can use the ZYCY10P3S2Tcells directly Cloning into Minicircle shRNA Parental Plasmids Linearize the MNSI vector with EcoRI BamHI Digest the plasmid overnight at 37 C Then purify the plasmid DNA Anneal the shRNA template oligonucleotides a Use regular non phosph
18. oo of the culture medium The pH should be around 7 and the ODso should be between 4 6 Record the ODeoo and the pH in your laboratory notebook in case you need to troubleshoot later a If the ODeo gt 8 and the pH lt 6 5 you must restart the protocol from the beginning Check the temperature and ventilation of the incubator b If the ODeoo is between 6 and 8 it is okay to proceed to the step 3 If the ODeoo was between 4 6 combine 200 ml of the overnight culture with 200 ml of 1X Induction medium a Add 20 ml of 10X Induction medium and 180 ml of sterile deionized water room temperature b The pH should now be between 6 9 and 7 4 If the pH is lt 6 9 add NaOH to adjust the pH to 7 If the ODeoo was between 6 8 combine 200 ml of the overnight culture with 400 ml of 1X Induction medium a Add 40 ml of 10X Induction medium and 360 ml of sterile deionized water room temperature b Divide the culture 600 ml total into two 1 liter sterile culture flasks 300 ml each c The pH should now be between 6 9 and 7 4 If the pH is lt 6 9 add NaOH to adjust the pH to 7 Incubate the mixture again at 30 C shaking at 250 rpm for 5 5 5 hours Longer induction times may increase bacterial death and cause genomic DNA contamination Take 1ml of the bacterial culture and do a miniprep followed by restriction digest analysis to check the quality of minicircle plasmid Pellet bacterial at 4 C and store the pellet at 20 C overnight Extra
19. or 30 seconds in a 42 C water bath Do not shake 6 Place cells on ice for 2 minutes C Recovery This procedure is very important for Kanamycin resistant constructs 1 Add 0 2 ml of room temperature S O C Medium to the vial Transfer to a bacterial culture tube 2 For tubes containing ligation reactions shake at 250 rpm 30 C or 37 C for 90 minutes 3 For tubes containing parental plasmid DNA shake at 250 rpm 30 C or 37 C for 60 minutes D Plating and Mini Preps 1 Pre warm culture plates to 37 C Chilled plates will decrease ZYCY10P3S2T E coli transformation efficiency 2 Spread 50 200 ul of bacterial solution on an LB plate containing 50 ug ml kanamycin Incubate overnight at 37 C 3 Keep the remaining solution at room temperature and spread onto a new plate the next day in case no colonies are seen 4 Pick 3 5 colonies to grow in 2 ml of LB containing 50 ug ml kanamycin Grow overnight and then extract the plasmid by miniprep 5 Check the minicircle parental plasmid by restriction digest analysis and sequencing 6 Ifthe parental plasmid looks correct make glycerol stocks from the miniprep bacterial culture and store them at 80 C You are now ready to produce minicircle DNA from the parental plasmid DNA E Growth and Induction of the Minicircle Producer Strain The following steps describe production of minicircle DNA from the parental minicircle plasmid which was produced in the previous steps Day 1 Inoculation
20. orylated oligos Dissolve the shRNA template oligonucleotides in an appropriate amount of deionized water to a final concentration of 20 UM b Set annealing reactions for each experimental shRNA template Page 4 ver 5 121016 www systembio com Minicircle DNA Technology Cat MNxxxA 1 1 ul Top strand shRNA template oligo 1 ul Bottom strand shRNA template oligo 18 ul 10 mM Tris HCl pH 8 5 20 ul Total Volume c Heat the reaction mix to 95 C for 2 min in a thermocycler d Turn off the thermocycler and let it cool to room temperature over 20 minutes 3 Ligate the shRNA template into the linearized minicircle shRNA parental plasmid a Setup a ligation reaction for each shRNA template 1 ul linearized vector 1 ul annealed double stranded shRNA template 1 pl 10x T4 DNA ligase buffer 6 ul deionized water 1 ul T4 DNA ligase 40 U ul 10 ul Total Volume b Incubate the ligation reaction at 16 C overnight 4 Propagation of the parental plasmid during cloning and screening can be accomplished with any strain of competent E coli 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual Minicircle Production Protocol The MC Easy Minicircle DNA Production kit provides a simple efficient way to produce high quality minicircle DNA The most common problems for producing Minicircle DNA are parental and genomic DNA contamination This kit offers an easy and reliable solution to eliminate gen
21. scription Multiple Cloning Sites MCS pMC BESPX MCS1 empty Spel EcoR1 Bglll EcoRV Xbal vector Sall Apal Spel EcoR1 Bglll EcoRV Xbal MC BESPX MCS2 t A pa Ei Nhel BstBl Swal BamHI Pstl Sacl Sall Apal paca open iesateccotene SV40polyA BstBl EcoRI Nhel Xbal Nhel Apol EcoRI PASO pMCEFL MCSSV4OpOWA sal Sami an pasta Moro owalteomtl SV40PolyA Swal BamHI ansizac PME CMY MCS EFL REP Xbal Nhel EcoRI BstBl SV40PolyA Swal BamHI pc o O am SV40PolyA Swal BamHI pass roa sam SV40PolyA Swal BamHI MN100B Page 20 ver 5 121016 www systembio com VII IX Minicircle DNA Technology Cat MNxxxA 1 Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site http www systembio com For additional information or technical assistance please call or email us at Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com System Biosciences SBI 265 North Whisman Rd Mountain View CA 94043 Licensing and Warranty Use of the Minicircle Technology i e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components of the Product to System Biosciences SBI within 7 calendar days Purchase and u
22. se of any part of the Product constitutes acceptance of the above terms The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product may not be resold modified for resale or used to manufacture commercial products without prior written consent of SBI This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research This Product shall be used by the purchaser for internal research purposes only and distribution is strictly prohibited without written permission by System Biosciences Limited Warranty SBI warrants that the Product meets the specifications described in the accompanying Product Analysis Certificate If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price SBI s liability does not extend to any damag
23. tional innovative method for degrading any contaminating genomic DNA using an ATP dependent DNAse reagent that will selectively remove genomic DNA but will ot affect minicircle DNA yield This method produces clean and effective minicircle every time http www systembio com minicircle dna vectors literature attR attB Promoter Tra n Ven e P Arabinose switches on OC31 integrase and Sce l Endonucklease genes S ES 32x Sce P Sites 32x Sce l Sites Promoter Parental Plasmid Bacterial Backbone KanR Degradation Page 2 ver 5 121016 www systembio com Minicircle DNA Technology Cat MNxxxA 1 Arabinose M 10kb 3kb B ZYCY10P3S2T E coli ZYCY10P3S2T E coli Minicircle producer competent cells have been prepared and tested by a modification of the procedure of Kay Kay MA He CY Chen ZY 2010 Nature Biotechnology These cells are suitable for the cloning of minicircle based plasmids ZYCY10P3S2T E coli minicircle producer cells are derived from a BW27783 bacterial strain that stably expresses a set of inducible minicricle assembly enzymes C31 integrase and l Scel homing endonuclease This bacterial strain produces purified minicircles in a time frame and quantity similar to those of routine plasmid DNA preparation making it feasible to use minincircles in place of plasmids in mammalian transgene expression studies Note Other competent E coli strains cannot be substituted
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