I DIA ACTSI - Indiana CTSI
Contents
1. 5 MATERIALS 5 1 Reagents 5 1 1 DNA Hydration Solution stored at room temp Qiagen Cat 158916 for manual DNA extractions only unless otherwise specified in SF 4 1 5 1 2 DI water from lab sink SOP SF 4 16 01 SOP FOR DNA QC py lo Page af 3 2a l N DIANA C T S STANDARD OPERATING PROCEDURE sal Trane une Senta Indiana CTSTSpecunen Storage Facility PL STANDARD OPERATING PROCEDURE FOR DNA QUALITY CONTRO CHAPTER 4 Specimen Processing HD Beton g Issue Date SOP 4 SF 16 01 I ftective Date 102 29 O SKPERSEDES SOP WA NI THORED BY Z lebete fanpfiti ODDATE _C of fk AIO Indiana C sf ssar Pechnician APPROVAT om te eee DATEL be CT eth et 7 Indiana C TSI SSF Director 3 A 7 QA APPROVAL a ae ll LEASE DAT tz E G 22 10 Quality Compliance Specialist 1 REVISION l1 Not Applicable inital version 2 PURPOSE 2 1 This Standard Operating Procedure SOP detines the procedures used in the Indiana CTS Specimen Storage Vacility SSP to ensure that Quality Control QC of DNA is performed in a compliant and uniform manner PRINCIPLE 4 Quality Control af DNA is performed bs various methods The SSP performs QC af DNA bs using a spectraphotometer emitting UV light and measuring absorbance at two different wavelengths to determine the concentration und purity 260 280 ratio of the DNA 4 SCOPL 4 1 This SOP applies to all SSF personnel performing QC of DNA It defin2. 4 Repeat the process outlined in steps 6 2 3 1 through 6 2 3 3 until the entire batch of samples is analyzed SOP SF 4 16 01 SOP FOR DNA QC Page 5 of 5 6 2 3 5 Remove the print out of the results and record on applicable processing worksheet 6 2 3 6 Make a photocopy of the print out and attach both the original and the photocopy to the applicable worksheet 7 REFERENCES 7 1 Nanodrop ND 1000 Spectrophotometer V3 3 Users Manual 7 1 1 Located in the SSF shared folder 7 1 2 Alternatively downloadable from the support section of the Nanodrop website http nanodrop com 7 2 Eppendorf BioPhotometer 6131 Users Manual 7 2 1 Located in the SSF shared folder 7 2 2 Alternatively downloadable from the support section of the Eppendorf website http www eppendorfna com 8 DOCUMENTATION 8 1 Records are maintained per SF 1 6 Controlled Document Management 8 2 Deviations are managed per the SF 1 9 Deviation Management SOP Of special note per scope of this SOP complying with investigator specific directives per SF 4 1 is not a deviation to this SOP but is noted on the applicable worksheet 9 APPENDICES 9 1 The current version of the following appendices are used to implement this SOP None DNA readings are placed with the applicable DNA extraction worksheet SOP SF 4 16 01 SOP FOR DNA QC
3. Page of 5 eee N D l A N A C 1 5 l STANDARD OPERATING PROCEDURE Clinical and Translational Sciences Institute Indiana CTSI Specimen Storage Facility TILE STANDARD OPERATING PROCEDURE FOR DNA QUALITY CONTROL CHAPTER 4 Specimen Processing Issue Date iO 99 10 Oe AE OA Effective Date 0 29 10 SOP SF 4 16 01 SUPERSEDES SOP N A AUTHORED BY APPROVAL QA APPROVAL 1 REVISION 1 1 Not Applicable initial version 2 PURPOSE 2 1 This Standard Operating Procedure SOP defines the procedures used in the Indiana CTSI Specimen Storage Facility SSF to ensure that Quality Control QC of DNA is performed in a compliant and uniform manner 3 PRINCIPLE 3 1 Quality Control of DNA is performed by various methods The SSF performs QC of DNA by using a spectrophotometer emitting UV light and measuring absorbance at two different wavelengths to determine the concentration and purity 260 280 ratio of the DNA 4 SCOPE 4 1 This SOP applies to all SSF personnel performing QC of DNA It defines the process of obtaining the concentration and the 260 280 ratio for DNA samples using the Eppendorf model Biophotometer 6131 and or the Nanodrop model ND 1000 spectrophotometers 4 2 All SSF processing SOPs may be superseded by specific directives from the investigator as directed in SF 4 1 Initial entry into the worksheet will define whether there are specific processing directives applicable to a specimen
4. any sample is less than 1 7 repeat the reading steps 6 1 2 3 through 6 1 2 6 for that sample 6 1 3 U SOP SF 4 16 01 SOP FOR DNA QC Page 4 of 5 6 1 3 2 1 If the 260 280 is still less than 1 7 refer to SSF Technical Advisor for advice Document explanations and or actions taken on applicable processing sheet 6 1 4 Naming saving and storing the data Short Term 6 1 4 1 Click the Show Report button on the menu screen 6 1 4 2 Click the Report name and type in the Run Identification number found on the applicable processing worksheet defined in 6 1 2 1 6 1 4 3 Click the Reports tab top left of window and use the drop down menu to Select Save Report 6 1 4 4 Click the Export Report Table Only in the new window that appears 6 1 4 4 1 Verify that the filename is the same as the Run Identification number listed in section 6 1 4 2 of this SOP 6 1 4 5 Save the text file in the Specimen Storage Facility NanoDrop data shared folder 6 1 4 6 Convert the text file Into an Excel document and save in the NanoDrop data shared folder 6 1 5 Naming saving and storing the data Long Term 6 1 5 1 Record QC data 260 280 ratio amp DNA concentration on applicable processing worksheet and or attach a printout of the Excel document to the applicable worksheet 6 2 Using the Eppendorf BioPhotometer 6131 Spectrophotometer for DNA QC 6 2 1 Sample Preparation 6 2 1 1 Prepare a BLANK by adding 60uL of DI water to a cuvette
5. compatible for use with the Eppendorf BioPhotometer 6131 using a pipette 6 2 1 2 Dilute each DNA sample by combining 2ul of DNA sample with 58uL of DI water into a cuvette 6 2 2 Machine Preparation 6 2 2 1 Press the Dilution button on the Eppendorf BioPhotometer 6 2 2 2 Enter the amount of DNA used in the dilution 002 on the key pad and press the Enter button 6 2 2 3 Enter in the amount of DI water used in the dilution 0058 on the key pad and press the Enter button 6 2 2 4 Pull the cuvette cover off of the biophotometer and set it aside 6 2 2 5 Insert the Blank sample cuvette prepped in section 6 2 1 1 above into the biophotometer in the proper orientation see Eppendorf BioPhotometer Users Manual and press the Blank button 6 2 2 6 Remove the Blank cuvette and discard it in an appropriate waste container upon completion of the analysis and the result has been printed out 6 2 3 Measuring DNA Samples for Concentration and Purity 6 2 3 1 Tap the cuvette gently on a hard surface a few times to evenly distribute the sample in the cuvette and to remove any bubbles within the sample 6 2 3 2 Insert a DNA sample cuvette prepped in section 6 2 1 2 above into the biophotometer in the proper orientation see Eppendorf BioPhotometer Users Manual and press the Sample button 6 2 3 3 Remove the sample cuvette and discard it in an appropriate waste container upon the completion of the analysis and the result has been printed out 6 2 3
6. es the process of obtaining the concentration and the 260 280 ratia tor DNA samples using the Fppendor mode Biophotometer 6 31 and or the Nanodrop model ND 1000 spectrophotometers 4 2 All SSP processing SOPs may be superseded by specific directives from the investigator as directed in SF 4 1 Ieitial entry into the worksheet will detine whether there are specitic processing directives applicable to a specimen S MATERIALS Sol Reagents SLI DNA Hydration Solution stored at room temp Qiagen Cat 158916 for manual DNA extractions only unless otherwise specified in SF 4 1 5 1 2 Di water from lab sink SOP SI 4 16 01 SOP POR DNA QC Page 2 of 5 5 1 3 FG3 Hydration Buffer stored at room temp Qiagen Cat 1023542 for DNA extractions via the Autogen Flexstar Method only 5 2 Supplies 5 2 1 Cuvette applicable for use with Eppendorf spectrophotometer if using the Eppendorf spectrophotometer 5 3 Equipment 5 3 1 Bar code Scanner optional 5 3 2 Nanodrop ND 1000 Spectrophotometer alternatively Eppendorf BioPhotometer 6131 Spectrophotometer appropriate for measuring absorbance at a wavelength range of 260 through 280 SF 3 10 and SF 3 11 5 3 3 Pipette s or other suitable dispensing unit capable of dispensing 2uL 60uL of a liquid sample SF 3 3 5 3 4 Vortex optional 6 PROCEDURE NOTE Record on the applicable extraction worksheet if exceptions to this SOP per SF 4 1 are per ap
7. lution if the DNA samples were extracted via the extracted via the manual Qiagen method reference SF 4 12 6 1 1 9 2 Use the FG3 Hydration Buffer if the DNA samples were extracted via the Auotogen Flexstar method reference SF 4 18 SOP SF 4 16 01 SOP FOR DNA QC Page 3 of 5 6 1 1 10 Clean the upper and lower pedestals when the Nanodrop finishes analyzing the sample 3 5 seconds 6 1 1 10 1 Raise the sample arm and clean the sample off both pedestals with a dry Kimwipe or similar product 6 1 1 11 Type the name of the solution used to blank the equipment in the software menu s Sample ID box 6 1 1 12 Again Load 2uL of the applicable Qiagen Hydration Solution used to rehydrate the DNA to the lower pedestal with a pipette close the sample arm and click the OK button 6 1 1 13 Clean the upper and lower pedestals when the Nanodrop finishes analyzing the sample 3 5 seconds 6 1 1 13 1 Raise the sample arm and clean the sample off both pedestals with a dry Kimwipe or similar product 6 1 1 14 View the resulting spectrum on the screen 6 1 1 14 1 The result should be a spectrum with a relatively flat baseline 6 1 1 14 1 1 If this is the case proceed to section 6 1 2 6 1 1 14 1 2 If the spectrum does not show a flat baseline repeat steps 6 1 1 9 6 1 1 14 until it does 6 1 1 14 1 3 If after several blanking attempts the spectrum stil does not show a flat baseline reference The Nanodrop User s Manual Trouble Sh
8. ooting sections 15 1 through 15 7 in SF 3 11 6 1 2 Measuring DNA samples for concentration and purity 6 1 2 1 Obtain DNA and applicable processing worksheet for recording values 6 1 2 1 1 Use SOP SF 4 12 DNA Purification Appendix A for DNA samples that have been manually extracted 6 1 2 1 2 Consult the SOP SF 4 18 processing worksheet for DNA extracted via the Autogen Flexstar method 6 1 2 2 Record the lot number and expiration date of the Hydration Solution used in step 6 1 1 9 6 1 2 3 Mix sample thoroughly prior to loading by gently inverting the sample tube several times or pipetting the sample up and down multiple times to mix or gently vortexing for a few seconds 6 1 2 4 In the software menu s Sample ID box scan using bar code scanner or type in the sample s ID 6 1 2 5 Load 2uL of sample onto the lower sample pedestal with a pipette close the sample arm and click the OKAY button 6 1 2 6 Clean the upper and lower pedestals when the Nanodrop finishes analyzing the sample 3 5 seconds 6 1 2 6 1 Raise the sample arm and clean the sample off both pedestals with a dry Kimwipe or similar product Repeat the process outlined in steps 6 1 2 3 through 6 1 2 6 for all samples until completion of the batch of samples have been analyzed 6 1 3 1 If there are any errors reference The Nanodrop User s Manual Trouble Shooting sections 15 1 through 15 7 in SF 3 11 and repeat the reading 6 1 3 2 If the 260 280 ratio for
9. plicable NOTE Store samples under conditions which sample is received per SOP SF 4 4 Sample Receipt Log in Tracking Resolve all discrepancies for sample receipt per SOP SF 4 4 6 1 Operation for the Nanodrop ND 1000 Spectrophotometer for DNA QC 6 1 1 Log in and machine preparation GLL a T2 6 1 1 3 6 1 1 4 NANO j et p i eh est 9 N O M 6 119 Ising IU login and password access the Nanodrop ND 1000 operating software installed on the computer connected to the instrument Double click on the Nanodrop icon to open the menu screen Click the Nucleic Acid button which prompts an instruction window to open reading Ensure sample pedestals are clean and then load a water sample After loading water sample click OK to initialize instrument Clean lower and upper pedestals on the Nanodrop by dropping distilled DI water on them and dry with a Kimwipe or similar product Load 2uL of DI water on the lower pedestal using a pipette Close the sample arm Click the OKAY button Clean the upper and lower pedestals when the Nanodrop finishes analyzing the sample 3 5 seconds 6 1 1 8 1 Raise the sample arm and clean the sample off both pedestals with a dry Kimwipe or similar product Load 2uL of the applicable Qiagen Hydration Solution that was used to rehydrate the DNA to the lower pedestal with a pipette close the sample arm and click the BLANK button 6 1 1 9 1 Use the DNA Hydration So
Download Pdf Manuals
Related Search