TITLE: Sample Cutting/Processing Protocol
AUTHORS: Khai-Minh H Nguyen, Katarina A Cohen, Abraham Palmer, Oksana Polesskaya

[DESCRIPTION]
Procedure for preparing a 96 sample plate for extraction on the EPmotion 5075 (can also do by hand). This protocol is created for Agencourt's DNAdvance extraction Kit.  

[BEFORE_START]
Make sure you have dry ice available (~2 pounds is good enough). 
All consumables and specific equipment are detailed in materials section.



[GUIDELINES]
Ensure you limit the amount of freeze-thaw cycles of samples. Aim to process half a plate (48 samples) in a day. It takes approximately 3-5 hours to do half a plate. 

[STEPS]
SECTION: Preparing Workstation
9. 1. Fill insulated foam box with dry ice and place empty tube rack to chill.

SECTION: Logging New Samples in Excel
1. 1. Place 16 samples on tube rack in two rows of 8.
Leave them to thaw for 15 min 
While you allow them to thaw, proceed to the next steps

SECTION: Processing Samples
13. Cut a small section of sample using your razor and place into corresponding well of the deepwell plate with your tweezers 
Spleen tissue = 5-6 mg
Tail = 8-10 mg 
NOTE - Maintain the same order in which you scanned your samples into the barcode processing sheet (Check well numbers A1, B1 C1)
Good "healthy" spleens should be intact/firm and have a vibrant deep red color. 
Jot down in the comments section of the corresponding well if the spleen looks particularly bad 
SECTION: Logging Processed Samples
18. Label new processed sample box (use cryo freezer box 2'', 89 slots) 
Palmer lab boxes are labelled as such: (PI Name) processed Box ## , Date, Initials of Lab Tech, Project 

SECTION: Logging New Samples in Excel
2. Download new Barcode Processing Template   

SECTION: Logging New Samples in Excel
4. In Column A "Origin", enter the name of the box in which the samples were shipped in. 

SECTION: Logging New Samples in Excel
5. Scan and enter transponder IDs 
Enter "no transponder" in comments column if transponder chip is not scanning. 
The first row of 8 samples will be entered into A1-H1
The second row of 8 samples will be entered into A2-H2 
Ensure the order is maintained throughout the protocol 
SECTION: Logging New Samples in Excel
6. Scan Barcodes 
Enter "no barcode" in comments column if barcode label is missing. 
Note the written code on tube if barcode label is missing 
Scan barcodes in the same order in which you scanned the transponders (ex. first row will be entered into A1-H1) 
SECTION: Logging New Samples in Excel
6.1. If barcode is a truncated version of the transponder ID, use the CONCACTENATE function 
e.g This will concatenate the first three zeroes "000" of the transponder ID to determine if it matches the scanned barcode ID. 
 

SECTION: Logging New Samples in Excel
7. Check for mismatches, missing transponder chips or barcodes. 
If "Match" Column shows as "FAIL" then mark sample as mismatch. 

SECTION: Preparing Workstation
10. Fill ice pan with ice and chill metal cutting blockon ice 

SECTION: Preparing Workstation
11. Add 50 µL of   to each well of the Deepwell plate (Catalog AB0932). 
Use reagent reservoir and a multichannel pipette to add lysis buffer to all wells
Place deepwell plate on ice . 
Write the DNA plate code (the code in which you named the file ex. John03) on the side of the plate
Write the date and your initials on the side of the plate 



SECTION: Preparing Workstation
12. Fill wash bottle with 70% EtOH. Clean tweezers, razor and metal cutting block with 70% EtOH and Kimwipes. 

SECTION: Processing Samples
16. Wipe gloved hands with RNAse Zap Wipes
SECTION: Processing Samples
15. Spray and wipe cutting block, razor and tweezers with 70% EtOH and Kimwipes
I recommend using a 100mL waste beaker to catch ethanol waste when spraying down the tweezers and razor


SECTION: Processing Samples
14. Return tissue to tube and onto the tube rack on dry ice. 
Ensure you maintain the order in which you scanned the samples. This will be important for keeping records of the location of the sample in the database. 
It is important that the tissue is immediately transferred onto dry ice when finished cutting. 
SECTION: Processing Samples
17. Repeat steps 13-16 until all 16 samples are completed 

SECTION: Logging Processed Samples
19. Place processed samples in labelled box while maintaining the order in which you scanned the samples

SECTION: Logging Processed Samples
20. Enter storage sample box name in column G and sample position in column H in the barcode processing excel sheet 
SECTION: Logging Processed Samples
21. Store processed samples in -80C Freezer 
SECTION: Lysis and DNA Isolation
24. Continue to EPmotion DNA Extraction Protocol if you have an EPmotion.
It is safe to store processed sample plate at -80 °C if you plan to continue a few days later. 

SECTION: Logging New Samples in Excel
8. Enter tissue type in column J
We typically extract from spleen or tail
SECTION: Database Entry
22. Create new extraction database on google sheets. Copy from the template below. Create another sheet for each new plate. 
Example Database
SECTION: Logging New Samples in Excel
3. Create a DNA plate code name
We typically do "PI Name ##"
Ex. John03 
This name will be assigned to this plate. Make sure you create a new name for each new plate. 
This will be the name that you will write on the following plates used in the protocol. 

Rename the newly downloaded Barcode processing template excel sheet with the DNA plate code name 
EX. John 03 Barcode Processing
SECTION: Database Entry
23. 
Column A - Enter DNA plate code (this is the name that you had written on the deepwell plate) ex. John03
Columns D and E - Copy and Paste Transponder ID's and Barcodes from the barcode processing excel sheet. 
Column K - Copy and Paste the Origin from the barcode processing excel sheet
Column M - Copy and Paste the Tissue Type from the barcode processing excel sheet
Column N and O - Copy and Paste the Storage Box of Sample and Position in Box 
Column Q - Copy and Paste any comments that you have put in the barcode processing excel sheet 
Column P - Enter freezer location of the processed sample box location
SECTION: Processing Samples
13.1. Cut along the blue line of the spleen.