Contents. Page 1. M13 Isolation Kit. EZgene TM

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1 Contents Table of Contents... 1 Introduction... 2 Storage and Stability... 2 Safety Information. 2 Kit Contents... 3 Before Starting... 3 M13 Isolation Protocol... 4 Trouble shooting guide... 6 Related products. 7 Limited use and warranty... 8 EZgene TM M13 Isolation Kit Page 1

2 Introduction EZgene TM M13 isolation kit allows rapid and reliable isolation of high-quality M13 phage DNA from appropriate host strain. The system combines the reversible binding properties of our matrix with the speed and versatility of spin column technology to yield approximately 3 to 10 μg M13 phage DNA from 1-3 ml of phage supernatant. In this procedure, the infected bacterial culture is centrifuged and Buffer PC is used to precipitate the phage particle supernatant and then the sample is loaded to the column. The specially designed matrix will allow the phage particles to remain intact on the surface of the matrix. The phage particles are then lysed and bounded to the matrix with the addition of Buffer PB. The pure M13 DNA is eluted with Elution Buffer or ddh 2 O while unwanted proteins and other impurities are washed away by DNA Wash Buffer. Storage and Stability All components are guaranteed for 12 months from the date of purchase when stored at C. Safety Information Buffer PB contains chaotropic salts, which may form reactive compounds when combines with bleach, Do not add bleach or acidic solutions directly to the preparation waste, ware gloves and protective eyewear when handling. Page 2 EZgene TM M13 Isolation Kit

3 Kit components Catalog# PD PD PD Preps DNA mini columns ml collection tube Buffer PB 1.5 ml 18 ml 80 ml Buffer PC 5 ml 80 ml 300 ml DNA Wash Buffer 2 ml 15 ml 3 x 24 ml Elution Buffer 1.5 ml 10 ml 25 ml User Manual Before Starting Exam this handbook and get familiar with each step. Prepare all components and have the necessary materials ready. PD : Add 8 ml absolute ethanol to DNA Wash Buffer before use. PD : Add 60 ml absolute ethanol to each DNA Wash Buffer before use. PD : Add 96 ml absolute ethanol to each DNA Wash Buffer before use. Centrifuge with swing-bucket rotor at room temperature capable of 10,000 rpm. EZgene TM M13 Isolation Kit Page 3

4 M13 Isolation Protocol (Spin) 1. Prepare 1~3 ml of infected M13 culture and incubate with vigorous shaking for 6-7 hours at 37 C. 2. Pellet bacterial by centrifuge at 5,000 rpm for 15 minutes at room temperature. 3. Transfer the clear supernatant into a clean tube. Be careful not to disturb the bacterial pellet during the transfer. 4. Repeat step Add 0.2 volume of Buffer PB and mix well by vortexing. Incubate at room temperature for 10 minutes. 6. Transfer 700 μl of the sample to the DNA column and centrifuge at 10,000 rpm for 30 seconds at room temperature. Discard the flow-through liquid and put the column back to the collection tube. 7. Repeat step 6 till the entire sample has been passed through the DNA column. Discard the flow-through liquid and collection tube. 8. Put the column into a new collection tube, add 600 μl of Buffer PC to the column and centrifuge immediately at 8,000 rpm for 30 seconds. Discard the flow-through liquid and put the column back to the collection tube. 9. Add another 600 μl of Buffer PC and incubate at room temperature for 1 minute. Centrifuge at 10,000 rpm for 30 seconds. Discard the flow-through liquid. Page 4 EZgene TM M13 Isolation Kit

5 10. Add 650 μl of DNA Wash Buffer and centrifuge for 30 seconds at 10,000 rpm. Discard the flow-through liquid. Repeat once. 11. Discard the flow-through liquid and collection tube, insert the column to a new collection tube. Centrifuge the column, with the lid open, for 1 minute at 12,000 rpm to remove any trace ethanol residues. Note: It is critical to remove the residual ethanol for optimized DNA yield. 12. Place the dried column in a clean 1.5 ml tube and add μl of Elution Buffer or ddh 2 O (Preheat at 60 C) to the center of the membrane and incubate at room temperature for 10 minutes. Note: Incubation at 60 C significantly increases the elution efficiency because the ssdna binds tightly to the membrane. 13. Centrifuge for 1 minute at top speed to elute the DNA. Yield and quality of DNA: Determine the absorbance of an appropriate dilution (20 to 50-fold) of the sample and read at OD 260 /OD 280. EZgene TM M13 Isolation Kit Page 5

6 Trouble shooting Guide Problems Possible reasons Suggestions Low DNA yield Incorrect host strain The host strain should carry the F -episome that is essential for M13 infection. Low DNA yield Bacterial culture overgrown or not fresh. Do not grow culture for over 7 hours. Low DNA yield Low ph of elution buffer Make sure that the ph of elution buffer is in the range of Low DNA yield Elution buffer added to the edge of membrane. Add the elution to the center of membrane and incubate for 10 minutes prior to elution. Low DNA yield Column clogged Use less than 3 ml of M13 phage supernatant per column. Avoid bacterial pellet during transfer. No DNA eluted Genomic DNA contamination. M13 DNA floats out of well while loading to agarose gel. Forgot to dilute the DNA was buffer with ethanol Carry over of bacterial cells during transfer the supernatant to column Ethanol residues eluted with the M13 DNA Add absolute ethanol as instructed. Make sure not to carry over the pellet when transfer the supernatant to the column. Spin the samples twice. Wash the column as instructed. Page 6 EZgene TM M13 Isolation Kit

7 Related EZgene TM Products Catalog # Product Name Preps Price $ PD HP plasmid mini kit PD HP plasmid mini kit PD HP plasmid midi kit PD HP plasmid midi kit PD HP plasmid maxi kit PD HP plasmid maxi kit PD BAC/PAC isolation kit PD BAC/PAC isolation kit PD BAC/PAC isolation kit 4x PD BAC/PAC isolation kit 20x PD ezfilter 96 BAC/PAC isolation kit 4x PD ezfilter 96 BAC/PAC isolation kit 20x PD M13 isolation kit PD M13 isolation kit PD ezfilter M13 isolation kit 4x PD ezfilter M13 isolation kit 20x EZgene TM M13 Isolation Kit Page 7

8 Limited Use and Warranty 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 Biomiga s 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 Biomiga. Biomiga s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be, at the option of Biomiga, to replace the products, Biomiga 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 technical support or learn more product information, please contact us at (858) or visit our website at Page 8 EZgene TM M13 Isolation Kit