Fungal Genomics Project Centre for Structural and Functional Genomics

DNA plate generation for sequencing: Template Group

STANDARD OPERATION PROCEDURE (SOP) Version 1.0 Effective Date: In Revision

Prepared by:

(signature on original document) S. Trosok, P. Pezeshki, J. Boleska

Date (yy.mm.dd):

09.04.03

Compiled by:

(signature on original document) S. Trosok, P. Pezeshki, J. Boleska

Date (yy.mm.dd):

09.04.03

Approved by:

(signature on original document) Project Head

Date (yy.mm.dd):

Report any changes to Aleks Spurmanis (aleks@vax2.concordia.ca)

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TABLE OF CONTENTS *** Please, do not write on this page ***

1 Scope & Application..........................................................................................................................................3 2 Summary of Method..........................................................................................................................................3 3 Introduction/Background.................................................................................................................................3 4 Responsibilities...................................................................................................................................................3 5 References...........................................................................................................................................................3 6 Safety Considerations........................................................................................................................................3 7 Materials/Equipment Required........................................................................................................................3 7.1 Equipment..............................................................................................................................................3 7.2 Material/Consumables...........................................................................................................................4 7.3 Chemicals...............................................................................................................................................4 8 Reagent Formulations........................................................................................................................................4 9 Procedures...........................................................................................................................................................4 9.1 DNA plate selection: ............................................................................................................................4

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Scope & Application

This procedure describes the methodology used by the DNA template group in the generation of 96-well DNA plates (to be sent for sequencing) using the Clone Tracking System.

Summary of Method
This procedure allows for the generation of 96-well DNA plates from 96-well miniprep template plates. The DNA plates are subsequently sent of for sequencing.

Introduction/Background
Available templates for DNA plate generation are obtainable on the Clone Tracking System. Based on the percentage of empty wells, DNA Plates are generated in a sequential fashion. The Clone Tracking System indicates the source and destination plates and well locations of all clones available for DNA plate generation. The Clone Tracking System must be updated prior to DNA plate generation. There are two types of DNA plate generations: 1) 96-well minipreps indicating >95% acceptable plasmid yields are directly transferred to 96-well DNA plates 2) 96-well minipreps indicating >5% unacceptable plasmid yields are used for primary or secondary empty filling, depending on the plasmid yield The goals of Empty Filling are to reduce the number of poor quality plasmids sent out for sequencing and consequently to reduce costs. The maximum poor quality plasmids allowed per 96-well plate has been set at 5%. Any value higher than this, then it is more cost-effective to take the trouble to Empty fill.

4 5 6 7
7.1

Responsibilities
P. Pezeshki, J. Boleska, M. Pineda, S. Trosok

References
1. Xxx

Safety Considerations
Not applicable.

Materials/Equipment Required
Equipment PC computer. Clone Tracking System program 0.5-10L multichannel pipetters (12 channels)

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111522408.doc 7.2 Material/Consumables 96-well culture plates 0.5-20 L tips Chemicals Not applicable.

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7.3

8 9
9.1

Reagent Formulations
Not applicable

Procedures
DNA plate selection: 1. To obtain available templates for DNA plate generation, go to the fungal genomics projects Clone Tracking Systems web site at https://fungalgenomics.concordia.ca/objclone/home.php. Provide the respective user name and password. 2. The Clone Tracking System indicates the available template plates which could be used for DNA plate generation. To generate DNA plates login to the Clone Tracking System. a) Click on Options, followed by Bar Coding, and select DNA Plate Generation. The System generates a filter screen which allows the user to choose a species and a template plate date range. This step is to shorten the length of the menu which will display the available template plates. b) Upon submitting the selected species/date range a new screen will indicate the templates to be used for primary filling (direct transfer) of the DNA plates. Select the template plates that will be used for the day as a set. Do this by clicking and dragging from the first to the last TMPc plate of the set. (Note: all selected plates will have the same DNA plate generation and send date). c) The DNA plate generation date must then be entered by clicking on the calendar icon and choosing the date in which the chosen set of templates will be used for DNA plate generation. d) In the location field enter the room no./ freezer no. in which the DNA plates of the chosen set will be stored e) If the date that the DNA plates are going to be sent to the sequencing centre is known, then enter the date in sent date field using the calendar icon, and click on submit button. If not then leave the sent and receive field empty and click on submit button. Note: The sent and receive date (the date that the result of the sequences for any particular DNA plate has put on the FTP server) can be updated using the update module on clone tracking system at a later date. However, if the sent date is known the receive date should be entered into clone tracking system when the sequence results are back.

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f) If the template plates selected at step (b) are full then proceed to step 9.1.1. If at least one of the templates selected at step (b) are not full then go to step (9.1.2). 9.1.1 Direct Transfer

i) Click on the submit button a new screen will open on the right side of the screen. ii) Fill the email address of the Sequencing Center person, and click on submit button again. Note: Upon submitting, the System generates a mapping file which coordinates the transferred template wells into the DNA plate wells including sequence IDs iii) Download mapping file by pressing the 'File Download' button at the bottom of the page. iv) Proceed to step 9.2.1: without empty filling 9.1.2 i) Empty filling

Click on the submit button, the System generates a table on the right side of the screen. Note: The table has columns which indicate 1) the number of empty wells in % 2) the well ids of the empty wells for ONLY the templates that have empty wells. The templates that do not have any empty wells are not shown on this screen. ii) Look in this table and decide whether any of the templates listed in the table needs an empty filling. Note: Normally the rule is, Any template plate which has > 5% empty wells will be empty filled. iii) At this point decide to do either: 1-choose not to fill the empty wells for a given DNA plate if the number of empty wells is less than 5% for all of the chosen templates Note: keep the page as default and continue to step (v) 2-fill the empty wells with clones from leftover templates or a newly prepped templates if the Number of Empty Wells is greater than 5%. Note: Select the plate(s) that will be used for Primary empty Filling by checking the box in the column labeled Empty Fill. Proceed to step (iv) 3- fill the empty wells with test clones by entering a comma separated list of empty well ids in each Test Clones field of the chosen template. Note: Fill email address field (this will inform McGill sequencing centre which well ids are test clones so that these sequences will be excluded from pipeline run). Proceed to step (v) iv) Select the secondary templates (leftover template or a fresh template) you would like to use for empty filling the primary templates selected in step(g.2) from the pull down menu in the right screen. Ensure that you click on Yes, I want Empty Fill Up

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Fill the email address of the Sequencing Center person. This person will receive DNA plate text files, one for each DNA plate created which lists the Sequence IDs and Well IDs of each plate. Note: Upon submitting, the System generates a mapping file which coordinates the transferred template wells into the DNA plate wells including sequence IDs vi) Download file by pressing the 'File Download' button at the bottom of the page, for future reference/record. vii) Verify that all entries are correct, and there is a Y indicating empty filling at the empty wells of the template in the Empty Filling column. Otherwise a N should be present viii) Proceed to step 9.2.2: with empty filling 9.2 Physical DNA Transfer 9.2.1 1. 2. Without Empty Filling

Using a multichannel pipetter, transfer 10 L of the thawed TMPc plate DNA in the same orientation to a labeled 96-well plate Seal and store to -20 C until scheduled date for sending. 9.2.2 With Empty Filling

1. Label a sterile tip box with the primary TMPc plate ID. 2. Using the printed version of the DNA plate generation file, prepare a tip mapping for the primary TMPc plate by removing tips corresponding to positions indicated by a Y in the empty filling column. 3. Using the mapped tip box, transfer 10 L of the thawed primary TMPc plate material in the same orientation. 4. Empty fill 10 L of the remaining wells with plasmid from the corresponding secondary TMPc plate. 5. Seal and store to -20 C until scheduled date for sending. Definitions / Terminology Primary TMPc plate (direct transfer): Template requiring empty filling indicated by N in the empty fill column of the mapping file generated. Secondary TMPc plate (empty filling): Template used to fill empty wells of the primary TMPc Plate indicated by a Y on the empty fill column of the mapping file generated. APPENDIX A (If required)

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