Bioline ISOLATE RNA Mini Kit, Product Manual

Page 1: ...ISOLATE RNA Kits Product Manual ISOLATE RNA Mini Kit ISOLATE Plant RNA Mini Kit...

Page 2: ...Product Manual www bioline com isolate 2...

Page 3: ...otic cells 09 7 3 Total RNA isolation from bacterial cells 10 8 Troubleshooting guide 12 ISOLATE Plant RNA Mini Kit 13 1 Kit contents 13 2 Description 14 3 Storage 14 4 Safety information 14 5 Product...

Page 4: ...sis Buffer R 6ml 30ml 125ml Wash Buffer AR 3ml 15ml 70ml Wash Buffer BR 2ml 8ml 40ml RNase free Water 1 5ml 6ml 2 x 15ml Spin Column R1 10 50 5 x 50 Spin Column R2 10 50 5 x 50 Collection Tube 50 5 x...

Page 5: ...is buffer also inactivates RNases thus protecting the released RNA The RNA is then bound to a silica membrane Subsequent wash steps remove the remaining cell debris Pure RNA is eluted in the final ste...

Page 6: ...00 x g 2 min 10 000 x g 2 min 10 000 x g 1 min 10 000 x g 1 min 10 000 x g 2 min 6000 x g 1 min Add 700 l Wash Buffer BR Discard filtrate Discard filtrate Discard filtrate Place Spin Column R2 in Elut...

Page 7: ...d in the Lysis Buffer the sample can be stored at 20 C for several months For information on how to work with RNA read Hints and Tips on page 19 1 Homogenize and lyse up to 20mg of tissue sample using...

Page 8: ...in Column R2 Centrifuge at 10 000 x g 12 000rpm for 1 minute Discard the filtrate and place Spin Column R2 in a new Collection Tube Note Ensure that ethanol has been added to Wash Buffer AR according...

Page 9: ...ll inhibit lysis of the cells and compromise the efficiency of RNA extraction Proceed directly to step 2 2 Add 450 l Lysis Buffer R to the sample Resuspend the sample completely by pipetting up and do...

Page 10: ...high concentration of RNA is required Increasing the volume of water will increase the yield but decrease the concentration of RNA Optionally perform a second elution step to increase the yield 9 The...

Page 11: ...ction Tube Transfer the remaining sample from step 5 to the same Spin Column R2 and centrifuge again at 10 000 x g 12 000rpm for 1 minute Discard the filtrate and place Spin Column R2 into a new Colle...

Page 12: ...e less than 20 l RNA degraded Inappropriate handling and storing of starting material Ensure proper handling and storage of samples Ensure that all steps are followed quickly RNase contamination Ensur...

Page 13: ...25ml Lysis Buffer BPR 6ml 30ml 125ml Wash Buffer APR 3ml 15ml 70ml Wash Buffer BPR 3ml 15ml 2 x 40ml RNase free Water 1 5ml 6ml 2 x 15ml Spin Column PR1 10 50 5 x 50 Spin Column PR2 10 50 5 x 50 Colle...

Page 14: ...ells by incubation in a chaotropic lysis buffer The lysis buffer also inactivates RNases thus protecting the released RNA The RNA is then bound to a silica membrane Subsequent wash steps remove the re...

Page 15: ...lation from plant tissue Transfer supernatant to Spin Column PR1 Homogenize and lyse tissue with 450 l Lysis Buffer APR or BPR Transfer to 1 5ml tube max speed 1 min 10 000 x g 2 min 10 000 x g 2 min...

Page 16: ...liquid nitrogen 1 1 Grind the sample to a fine powder using a mortar and pestle in the presence of liquid nitrogen Take care that the sample does not thaw during or after grinding 1 2 Transfer the sam...

Page 17: ...Spin Column PR2 Centrifuge at 10 000 x g 12 000rpm for 1 minute Discard the filtrate and place Spin Column PR2 in a new Collection Tube Note Ensure that ethanol has been added to Wash Buffer AR accord...

Page 18: ...e less than 20 l Inappropriate handling and storing of starting material Ensure proper handling and storage of samples Ensure that all steps are followed quickly RNA degraded RNase contamination Ensur...

Page 19: ...y impossible For example autoclaving a solution containing bacteria will destroy the bacterial cells but not the RNases released from the cells 2 Sources of RNase Skin The presence of RNases on human...

Page 20: ...stidine modification of the bases If DEPC treated water is made in house always remember to autoclave before use to degrade the DEPC RNase inhibitors The use of RNase inhibitors is highly recommended...

Page 21: ...tracted from see table on page 21 In general for good quality RNA the bands should be distinct with no smearing underneath them and the 28S band larger should be approximately twice as intense as the...

Page 22: ...18S 1 9 23S 2 9 25S 3 7 Yeast S cerevisiae 18S 2 0 26S 3 8 E coli 16S 1 5 23S 2 9 Xenopus 18S 1 8 28S 4 0 Worm C elegans 18S 1 7 28S 3 5 b Technical SUPPORT For technical assistance or more informati...

Page 23: ...ches BIO 37105 DEPC treated Water 10 x 10ml BIO 38030 10x MOPS EDTA NA Acetate Buffer 1 Litre BIO 38027 Elite Human HEK293 Total RNA 100 g BIO 38034 Elite Human HeLa Total RNA 100 g BIO 38035 Elite Mo...

Page 24: ...giepark TGZ 2 D 14943 Luckenwalde Tel 49 0 3371 68 12 29 Fax 49 0 3371 68 12 44 email info de bioline com Australia Bioline Aust Pty Ltd PO Box 122 Alexandria NSW 1435 Tel 61 0 2 9209 4180 Fax 61 0 2...