T510002-T510008 Dicer Enzyme Kit
Contents
1. METHODS AND PROCEDURES Preliminary Notes 1 We recommend that you use dsRNA templates of 500 1000 bp in length The dicer enzyme might not digest well if the dsRNA is smaller than 300 bp 2 Double stranded RNA generated using the TurboScript T7 RNA Transcription Kit can be used directly for the Recombinant Dicer Enzyme Kit without purification However dsRNA purified using standard lithium chloride purification procedures can give slightly better results 3 For first time users we strongly recommend using the Dicer siRNA Generation Kit Cat T510001 Generation of siRNA Using Recombinant Dicer Enzyme 1 Keep the Dicer Reaction Buffer at room temperature while assembling the reaction The following amounts are for a single 10 ul reaction 2 5 ul x Nuclease free water x ul dsRNA 1 ug 1 ul 10 mM ATP 0 5 ul 50 mM MgCl 4 ul Dicer Reaction Buffer 2 ul Recombinant Dicer Enzyme 1 Unit NOTE ug of dsRNA control template will yield about 0 5 ug of d siRNA which is sufficient for one or two transfections in 24 well plates using the GeneSilencer siRNA Transfection Reagent Adjust the reaction volume accordingly if you need more d siRNAs IMPORTANT Avoid using excess recombinant dicer enzyme as it may decrease the amount of diced siRNA dsiRNA that can be generated from the digestion reaction 2 Incubate overnight 12 to 18 hours at 37 C 3 Stop the reaction by adding 2 ul Dicer Stop Solution 4 Ch2. Z Genlantis A division of Gene Therapy Systems Inc Recombinant Human Dicer Enzyme Kit PRODUCT SUMMARY Cat No T510002 50 Units T510008 4 x 50 Units Description The Recombinant Human Dicer Enzyme Kit uses an ultra active form of human recombinant dicer enzyme that can cleave more than 95 of dsRNA template into 22 bp siRNAs within 12 hours under optimized reaction conditions Figure 1 This provides sufficient siRNAs in every single species to achieve effective gene silencing Fig 1 Efficient Digestion of Double Stranded RNA with Recombinant Human Dicer Enzyme 1 ug of a 700 bp dsRNA after 12 hour of digestion by the dicer enzyme Lanes 1 marker 2 1 ul dicer 3 1 5 ul dicer 4 2 ul dicer 5 2 5 ul dicer 6 3 ul dicer 7 3 5 ul dicer 8 4 ul dicer 9 1 ug dsRNA control Please note The Recombinant Human Dicer Enzyme Kit is intended FOR RESEARCH USE ONLY Components Enzyme 0 5 unit ul 4 tubes x 100 ul Cat 1510008 4 tubes x 200 ul Cat T510008 4 tubes x 25 ul Cat 1510008 4 tubes x 50 ul Cat 1510008 4 tubes x 100 ul Cat T510008 Nuclease free Water 1 tube x 1 ml Cat T510002 Nessie anise mi Cat 1000 Recombinant Human Dicer Enzyme Kit Page 1 of 2 Shipping and Storage The Recombinant Human Dicer Enzyme Kit is shipped frozen For maximum stability and long term use immediately store at 20 C upon receipt All components are stable for six months when stored properly
3. 1 5 ml For 3 minute transformation into E coli TurboCells Chemically Competent E coli 20 x 50 ul C300020 TurboCells F Chemically Competent E coli 20 x 50 ul C301020 Recombinant Human Dicer Enzyme Kit Page 2 of 2 Please note The Recombinant Human Dicer Enzyme Kit is intended FOR RESEARCH USE ONLY Limited Label License for the Recombinant Dicer Enzyme This product is covered by several patent applications owned by the Stanford University The purchase of this product conveys to the buyer the limited non exclusive non transferable right without the right to resell repackage or further sublicense under these patent rights to perform the siRNA production methods claimed in those patent applications for research purposes solely in conjunction with this product No other license is granted to the buyer whether expressly by implication by estoppel or otherwise In particular the purchase of this product does not include nor carry any right or license to use develop or otherwise exploit this product commercially and no rights are conveyed to the buyer to use the product or components of the product for any other purposes including without limitation provision of services to a third party generation of commercial databases or clinical diagnostics or therapeutics In addition any user that purchases more than 5 000 in any calendar quarter may be outside the above research license and will contact Stanford Universit
4. eck d siRNA 22 base pairs by using one of the following methods a 3 agarose Gel TAE b 15 native polyacrylamide gel 29 1 cast in 1X and electrophoresed in 0 5X TBE Run at 10 Watts and 4 C Visualize RNA by staining with ethidium bromide Version JL12212002 TROUBLESHOOTING GUIDE Possible Causes Recommended Solutions d siRNA yield is low quality No dsRNA or poor dsRNA Check the amount of dsRNA added Make sure that the correct fractions from the RNA Purification Columns are used and or purify dsRNA before dicer reaction Too much Recombinant Use 1 unit of the enzyme for every microgram Dicer Enzyme was added of dsRNA 10 mM ATP 1s old Use fresh ATP solution RELATED PRODUCTS Product Cat No For efficient generation of siRNA Dicer siRNA Generation Kit 5 genes 50 transfections T510001 TurboScript T7 Transcription Kit 20 Reactions T510003 RNA Purification Column 1 20 Columns T510004 RNA Purification Column 2 20 Columns T510005 For efficient and functional siRNA transfection GeneSilencer siRNA Transfection Reagent 200 reactions 0 75 ml T500750 GeneSilencer siRNA Transfection Reagent 5 x 200 reactions 5 x 0 75 ml T505750 For efficient transfection of DNA into diverse cell lines GenePORTER 2 Transfection Reagent 1202007 75 reactions 0 75 ml GenePORTER 2 Transfection Reagent 1202015 150 reactions 1 5 ml GenePORTER 2 Transfection Reagent 1202075 750 reactions 5 x
5. y for a license This product is sold pursuant to a license from Stanford University and Stanford University reserves all other rights under these patent rights For information on a license to the patent rights for uses other than in conjunction with this product or to use this product for purposes other than research please contact Stanford University at 650 723 0651 This is Stanford University reference S02 028 Please contact us for a complete list of gene therapy research products Genlantis A Division of Gene Therapy Systems Inc 10190 Telesis Court San Diego CA 92121 Tel 858 457 1919 888 428 0558 Fax 858 623 9494 Website http www genetherapysystems com Version JL12212002
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