CTR:Protocols

Overview

Replace this sentence with a brief description of the protocol and its goal. To be edited!

Beckman Sequencing

• PCR product: 20μL. Primer: 25μL of 3μM
• Use Neptune full-skirted 96-well PCR plates.
• Seal plate with foil adhesive PCR sealing film.

1. Go to https://psf.beckmangenomics.com/ and login
2. Select "Create New Project" at top left
3. Select "Single Pass PCR Sequencing" under High Throughput Sequencing - Other
4. Enter "Project Name" (distinct to this plate, e.g. Lori_Africa09_1)
5. Enter how many plates of PCR products you are sending under "Sample Format"
6. Enter "Amplicon size"
7. Select No under "Are your samples purified"
8. Fill out "Sequencing Requirements" (e.g. if you are sending one plate with two directions, total will be 192)
9. Click Next
10. Download the template under "Primer Information" ("singlepasspcr_primer_template.xls")
11. Under "Sample Information" enter plate name, amount (20μL) & amplicon size. Under "Primer Information" enter plate name, amount (25μL), & conc. (3μM)
12. Upload the template by clicking "Attach File"
13. Click Next
14. Next page is pretty self explanatory. Choose ".ab1" under "Data Delivery"
15. Click Next
16. Enter Purchase Order # info (on back of computer room door)
17. Click Next
18. Confirmation. You will need to include this page with the shipment.

To Ship:

1. Login to FedEx account
2. Create Shipment
3. Under "Company" in the "TO" fields, start typing Beckman. The address should pop up. if not:

Beckman Coulter Genomics
Attn: Genomic Services
36 CHERRY HILL DR
DANVERS, MA 019232575
(800)361-7780

1. Choose Standard Overnight in the "Service Type" under "3. Package and shipment details"
2. Ball park estimate the weight with dry ice
3. Select Your Packaging
4. On the right hand side, Click "Edit" by "Special Services"
5. Select Dry Ice and Deliver without Signature under "Signature type"
6. Click Ship at bottom of page
7. Confirm Details and Ship
8. Print Shipping Label
9. Pack up box with plates and dry ice (buffer plates a little so they don't shatter). Be sure to fill out and include the dry ice label on the box (available at the storeroom). Stick this on LAST, after the box has been sealed.
10. Last pickup at storeroom is around 3:30pm

Notes:

• For getting dry ice from the store room, remember to fill out the card with the recharge ID. Only whole numbers in pounds are accepted. For example, you can get 1 or 2 pounds of dry ice, but not 1.5.
• To ship with dry ice, remember that the styrofoam box with your contents must be in a cardboard box.
• Remember to deduct the total from the signup sheet on the computer room door.

Sequencing at the Core

1. Get EXO, SAP, and Dilution Buffer tubes from “EXO SAP” box in mini freezer. Keep EXO and SAP on ice to prevent enzymes from denaturing.
2. Flick or pipette mix EXO and SAP – do not vortex. Make up master mix:
EXOSAP Master Mix (1 reaction in 1 direction)

Reagents	1x
dH2O	1μL
Dilution Buffer	0.25μL
EXO	0.25μL
SAP	0.5μL
PCR Product	3.0μL
TOTAL	5.0μL

3. Aliquot 2uL master mix into PCR tubes or 96-well PCR plate.
4. Combine with 3uL PCR product per tube/well. Pipette mix.
5. Spin down tubes/plate and run EXO SAP program on PCR machine (~1hr):

• 37°C for 30 min (to perform digestion)
  
• 80°C for 10 min (inactivate the enzymes)
  
• 4°C forever
  

6. Store cleaned PCR product in freezer (-20) until ready to set up BIG DYE reaction.

BIG DYE:
1. Get Big Dye from mini freezer. Keep on ice and cover while thawing (light sensitive).
2. Get Seq Saver from refrigerator.
3. Make up master mix:
BIG DYE Master Mix (1 reaction in 1 direction)

Reagents	1x
dH2O	3μL
Seq Saver	0.5μL
Big Dye (Light Sensitive!)	0.5μL
Primer (3μM)	1.0μL
Clean PCR Product	5.0μL
TOTAL	10.0μL

• The volume of PCR product can vary from 1.5-5ul depending on how concentrated your PCR product is. Remember to adjust the volume of water if you adjust the volume of PCR product.
  

4. Aliquot 5uL master mix into PCR tubes or 96-well PCR plate.
5. Combine with 5uL PCR product per tube/well. Pipette mix contents.
6. Spin down tubes/plate and run BIGDYE05 program on PCR machine:

• 96 degrees for 10 sec
  
• 96 degrees for 10 sec
  
• 50 degrees for 20 sec
  
• 60 degrees for 4 min
  
  • 35 cycles of steps 2-4
• 4 degrees forever
  

7. Store cycle sequencing product in freezer (-20) until ready to send to Core.

Sequencing sign up

1. Sign up to sequence at Genoseq CORE.
2. Click on R2R Signup (list) (found on the left hand side) and fill out required information.
3. Answer YES for request cleaning service.
4. Bring the given reference number with you, check in your samples when you drop them off, and bring back sample racks. Alternatively, you can bring your samples over to the Core and sign up there.

• Carry samples on ice to the Core.
  

BioAnalyzer

1. Quantify samples (use Nanodrop, Qubit, or PicoGreen) and record concentration.
2. Using dH2O, dilute 1ul of sample to ~ 2.5-3 ng/uL. If using Nanodrop numbers, use 5-10 ng/uL to account for error. The total final volume must be at least 3ul.
3. Take samples on ice to Core (CHS 36-125). Sign up on the sheet and place samples on a rack. Label the rack and store in freezer.
4. To check for results, log into WebSeq and click "Genotyping" on the left side menu.

Notes:

• Maximum number of samples for DNA High Sensitivity and RNA Pico chips is 11. Other chips are 12 samples.
• Try to fill up chip as much as possible by coordinating with other labs.

Printing field labels

1. Create Excel sheet listing all field numbers. Save as 1997-2003 Excel format and transfer to flash drive or send in email.
2. On Nanodrop computer, log on to "General" account.
3. Open BarTender program. Open an older file to edit as a template or start a new one.
4. Go to "Database Connection Setup". Select the Excel sheet with the field numbers.
5. Edit the label as necessary.
6. Print label.
   1. First click "Test Print". This will print the first entry in the Excel sheet.
   2. If the label looks fine, enter the field number range in "Selected Records" (for example, 1-200). Recommend printing no more than 100-200 at a time to ease organization and to check for toner fading.
7. Save the file.

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