OPTIMIZE YOUR SAMPLE PREP PROTOCOL Precellys Webinar Thursday 11th 2015
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1 OPTIMIZE YOUR SAMPLE PREP PROTOCOL Precellys Webinar Thursday 11th 2015
2 WEBINAR ORGANIZATION & RULES 2 1) Your microphone will be muted during the presentation to minimize the interferences 2) If you have questions or remarks during the presentation, please write it in the questions tab. After the presentation, the speakers will answer to your questions.
3 AGENDA 3 1. General information about Bead-beating 1. Precellys products 2. Sample prep optimization tips 1. Kits selection 2. Online tool: Application Center 3. Application & case study 4. Conclusion
4 GENERAL ADVANTAGES OF BEAD-BEATING 4 Same 3D motion applied to all tubes REPRODUCIBILITY THROUGHPUT 24 x 2/0.5mL 12 x 7mL 6 x 15mL BEAD- BEATING CROSS- CONTAMINATION EFFICIENCY Single use prefilled tubes with beads High speed up to rpm
5 PRECELLYS FAMILY OF PRODUCTS A COMPLETE HOMOGENIZATION SOLUTION 5 Multiple applications High throughput and flexibility High throughput Routine work Flexible Affordable Cooling option Protects sensitive molecules 27 different lysing kits, Empty tubes & beads in bulk
6 OPTIMIZATION TIPS
7 PRECELLYS LYSING KITS A COMPLETE RANGE 7 4 Volumes: 0.5, 2, 7 & 15mL Soft Tissue Hard Tissue Elastic Tissue Microorganism Liver Brain Leaf Organs Etc. Hair Bones Tumor Corn Etc. Skin Cartilage Innervated tissue Etc. Yeast Bacteria Viruses Spores Etc. CERAMIC METAL CERAMIC MIX Une société GLASS du Groupe
8 CHOOSING THE RIGHT KITS 8 What is your sample size/weight, what is your buffer volume? Less than 20 mg (i.e. biopsies) and between µl choose 0.5mL tubes Between mg and between µl choose 2mL tubes Between 200 mg 2 grams and between 1200 µl 5mL choose 7mL tubes Between 2 grams 5 grams and between 5mL 10mL choose 15mL tubes What is your downstream application? DNA, RNA, protein, enzymes, metabolite extraction? For thermo-sensitive applications such as RNA, enzymatic, and metabolite extraction Cryolys is needed
9 APPLICATION CENTER 9
10 R&D TOOL FOR ROUTINE LAB WORK 10 One sample type Soft tissue Microorganisms Hard tissue Write protocol into SOP Routine Precellys Several protocols = same results Ceramic or mix 0.5 or 0.1mm Glass Large ceramic or metal Optimiza -tion Speed, cycle, time, break
11 11 APPLICATION & CASE STUDY
12 CASE STUDY: HOMOGENIZING TUMOR TISSUE 12 Research Aim: Homogenize mouse tumor tissue from different organs for RNA extraction Sample type and lysing kits Breast tumor xenografts and lymph node metastases / CK14 2mL tubes with 600 µl lysis buffer Precellys24 protocol 5500 rpm, cycle number variable (Fig 1) Results The presence of 28S and 18S RNA ribosomal subunits represents high quality, non-degraded RNA Figure 1. Ten Precellys protocols tested for tumor homogenization 28 S 18 S CK14 2mL: 1.4mm ceramic beads Figure 2: Total RNA run on an electrophoresis gel after Precellys homogenization using the ten tested protocols
13 CASE STUDY: RICE GRINDING 13 Research Aim: Grind rice for DNA extraction to meet quality control guidelines in the food industry Sample type and lysing kits Dry grinding 3 rice grains species (Orysa sartiva L.)/ MK28-R 2mL Precellys24 protocol 6000 rpm, 2x30 sec (20 sec break) Results Total DNA was loaded onto a gel after Precellys grinding to check DNA quality and quantity clear, sharp bands were observed Figure 1. The Precellys24 pulverizes 3 different rice grains in a minute Precellys 24 M P1 P2 P3 P4 M1 M Figure 2. DNA extraction efficiency was confirmed using PCR amplification of a gene-specific rice marker. MK28-R 2mL: 2.8mm metal beads in reinforced tubes
14 CASE STUDY: BACTERIAL LYSIS 14 Research Aim: Lyse E. Coli for soluble protein purification Sample type and lysing kits E. coli cells normalized to an OD 600 = 50 / 1mL of culture into a VK01 2mL Precellys Evolution protocol 9000 rpm, 6x30 sec in the cold room (60 sec break) Results The protein extracts were separated on SDS-PAGE, followed by Coomassie blue staining, which shows high protein extraction efficiency and the presence of strong bands VK01 2mL : 0.1mm glass beads Figure 1. Total soluble protein profiles after Precellys homogenization using VK01 glass beads (Lanes 2 and 3)
15 Response (Cts/ g) CASE STUDY: WHOLE IRIS-CILIARY BODY PORK LYSIS 15 The Minilys has a max speed of 5000 rpm, use stronger lysing matrix CKMix50-R 2mL, 7mL or CK68-R 2mL, 15mL homogenize whole organs, hair, etc. Sample type and lysing kits Whole iris-ciliary body pork (0.7 grams) / CKMix50 7mL with 1.4 ml of NaCl solution Homogenization of whole iris-ciliary body into 7mL vial with Minilys Minilys protocol 5000 rpm, 4x30 sec (15 sec break) Results 1,00E+07 1,00E+06 1,00E+05 Precellys Average Minilys The analyte levels are equivalent between the Minilys and Precellys. By using the right lysing kit, the Minilys is effective in homogenizing harder tissues 1,00E+04 1,00E+03 1,00E+02 Avera 1,00E+01 1,00E+00 Precellys - E1E2 Minilys - E1e2 Figure 1. LC-MS/MS analysis of the target analyte after Precellys and Minilys homogenization CKMix50 7mL : 2.8 and 5.0mm ceramic beads
16 CASE STUDY: EXTRACT VIABLE BACTERIA 16 Sample type and Precellys lysing kit Skin (122 mg) and spleen (39 mg) / CK14 and CK28 2mL with 1mL of 0.7% sterile saline inoculated with S. pyogenes Precellys24 protocol 6000 rpm, 1x30 sec (spleen) 6000 rpm, 2x30 sec (skin) Other Applications Cell fractionation Mitochondrial isolation from cell lines Minilys protocol: 3000 rpm, 1x10 sec with CK14 2mL Figure 1. Precellys homogenization has no effect on the viability of S. pyogenes after short or long homogenization times. Viability was assessed by cultural method and compared to a known inoculum concentration (CFU/mL), added to each tissue sample. Single cell dissociation from tissue Precellys Evolution protocol: 4600 rpm, 2x10 sec with 1-2 CK28, CK50 or CK68 beads (dependent on tissue size)
17 CONCLUSION 17 Complete Precellys Lysing Kit range for any sample type animal tissue, microorganisms, plants, bone, hair Precellys Homogenizers can be used for R&D and routine lab work An Application Center with >2000 publications and application notes
18 CONTACT US 18 >> Request your free Precellys demo >> Request your free demo kits >> Ask for application support >> Find your local distributor
19 19 ALWAYS VALID Write an Application Note and get 100 free preps (2 lysing kits) Terms and Conditions 1. Mention " STAR SHAREPROT" in your order / 2. Incentive without time limitation / preps means 2 lysing kits of 2 or 7ml dependiong on the samples / 4. For any detail, contact your Sales Area Manager.
20 THANK YOU FOR YOUR ATTENTION Feel free to ask any question with chat box?
21 THANK YOU FOR YOUR ATTENTION 21 1) Use the questions tab if you have questions or remarks. 2) Please take 2 minutes to fill out the survey (only 5 questions) to continue the improvement of our next webinar and help us to define the next subjects. 3) The presentation will be put online you will receive an giving the download link
22 22 APPENDIX
23 25 LYSING KITS TO COVER ANY APPLICATION 23 Samples Kit Name Beads Size (ml) MK28/MK28-R 2.8mm stainless steel 2 CK28/CK28-R 2.8mm ceramic 2, 7, 15 Hard tissues Soft tissues CK68-R 6.8 mm ceramic 2, 15 CKMix50-R 2.8 mm/5.0 mm ceramic 2, 7 GK60-R 0.7mm garnet flakes + 6mm ceramic bead CK14 1.4mm ceramic 0.5, 2, 7 CKMix 1.4 mm/2.8 mm ceramic 2 2 VK05 0.5mm glass 0.5, 2, 7 Micro-organisms VK01 0.1mm glass 2 VK01/VK mm glass/0.5 mm glass 7 CK01 0.1mm ceramic 2, 7, 15 Soil grinding SK38 Mix glass and ceramic 2, 7
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