CTR:Protocols

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Lab Recipes

Feather Extraction Protocol

Smith Lab Feather Extraction Protocol

Smith Lab Feather Extraction Protocol Using Phenol Chloroform

Rhodopsin Amplification Protocol

Rho Amp PCR

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.

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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	1.0μL
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	3.0μL
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°C for 10 sec
  
• For 35 cycles:
  • 96°C for 10 sec
    
  • 50°C for 20 sec
    
  • 60°C for 4 min
    
• 4°C forever
  

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

Sequencing sign up

1. Sign up to sequence on WebSeq.
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.

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Qubit

• Before starting, take Qubit HS (for low-concentration samples up to 100ng/μL) or BR kit (for samples 2-1000ng/μL) from fridge and thaw buffer and dye to room temperature.
• The dye is light-sensitive; make sure it is wrapped in foil and/or covered.
• Use Qubit assay tubes only (we have 0.65mL Axygen tubes).
  

Sample Preparation
1. Make master mix of buffer and dye in the ratio of 200μL buffer : 1μL dye per sample. Take into account the number of samples you have and the 2 standards, and add extra for error.
2. Make STD-1 by adding 200μL of mix to a tube (1μL dye and 199μL buffer). This is the blank.
3. Make STD-2 by adding 190μL of mix and 10μL of standard to a tube.

• For HS kit, use the 10ng/μL standard (100ng).
• For BR kit, use the 100ng/μl standard (1000ng).

4. For all other samples, add 199μL mix to each tube, then add 1μL DNA sample. This can be adjusted to 198μL mix and 2μL DNA - you will just have to account for the difference when you do the concentration calculation.

Qubit fluorometer

• Fluorometer can be found either at the Core ($5 charge) or in the Alfaro lab on the 2nd floor.

1. Turn on machine either by plugging it in or tapping the screen. Choose the appropriate program.
2. To blank machine, place STD-1 tube into machine, close lid, and press measure.
3. Place STD-2 tube into machine, close lid, and press measure.
4. Record the reading for STD-2. It should be around 500 for HS or 5 for BR.
5. Remove STD-2 and begin measuring and recording readings for all other samples.
6. At the end of process, measure STD-2 again. Reading should still be around 500 for HS or 5 for BR. If it is not, start the process over again (re-read standards and DNA tubes, etc.)

Calculation
Concentration of sample = QF x (200/volume of DNA used)

• QF is the value given by the fluorometer
• Remember to look at the units given by the fluorometer and take that into account when making your final calculation.

Shortcut using 1μL of DNA:

• HS: QF / 5 = ____ ng/μL
• BR: QF x 200 = ____ ng/μL

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PicoGreen

This is similar to the Qubit but uses the PicoGreen kit and Victor plate reader instead. Again, the dye is light-sensitive, so make sure it is wrapped in foil or covered.

Make sure 1x TE and standards are prepared. Prepared 1x TE should be above the PCR machines and the standards should be in the fridge. If they are out:

• Dilute the concentrated (20x) buffer from the kit 20-fold with dH₂O.
  
  • For example: 20x buffer * x = 1x buffer * 200mL
    
  • x = 10mL concentrated buffer. Add 190mL dH₂O.
• Prepare standards (makes 50μL):

μL stock standard	μL 1x TE	ng/μL
50	0	100
25	25	50
5	45	10
2.5	47.5	5
0	50	0

• • Can use fewer standards or make additional standards with smaller concentrations if desired.
  • Store leftover standards in the fridge.

To prepare DNA samples in plate:

1. Take dye and prepared standards from fridge and let them thaw to room temperature.
2. Make master mix of 1x TE and dye in the ratio of 200μL TE : 1μL dye per sample. Take into account the number of samples you have and the number of standards, and add extra for error.
   1. This can be poured into a reservoir if using a multichannel pipet. Remember to keep it covered.
3. Obtain black PerkinElmer 96 well plate.
4. Add 1-2μL of each standard in a row. Add the same volume of DNA sample into the remaining wells.
   1. The Victor machine reads plates horizontally across rows, not down each column.
5. Add 198-200μL of TE/dye mix to each well and pipet mix.
6. Incubate plate for at least 5 minutes in the dark.

To read and calculate sample concentrations:

1. Place plate in Victor plate reader machine.
2. Open Wallac Victor 3 program.
3. Click the second button at the top, "Define plate map and start...". Choose your program.
   1. You may have to create your own folder and copy or customize a PicoGreen program from another folder.
4. Define which wells on the plate are measured vs. empty by clicking on individual wells and/or selecting across rows. Start the program.
5. After the plate is done reading, click the first button at the top, "Explore protocols and results". A screen should show up with a list of all the plates run under the program you ran.
6. Double click the file you ran. Go to File > Export. Save the document as an excel file on a flash drive and eject.
   1. Do not name the file with any periods. It will interpret text after periods as the file extension.
7. On another computer, open the Excel file. Go to the first sheet.
8. In the last column, you will see PicoGreen values. These must be extrapolated to concentrations:
   1. Next to the values of the standards, enter the standard concentrations (0-50).
   2. Insert a scatter plot. Right click the box and click "Select Data". Under "Legend Entries", click the "Add" button. For Series X values, select the standard concentrations you entered. For Series Y values, select the PicoGreen values that the standards correspond with and press OK. A scatter plot should show up with the points in a relatively straight line.
   3. Click on the points on the graph to select all of them. Right click and select "Add trendline". Also click the options to show equation and R². These should now show up on the graph. The R² value should be close to 1.
   4. In a new column, enter the formula to calculate the concentration of each sample. For example, if your equation is y = 500x + 1000, the formula in the cell should look like " =(H2-1000)/500 ". This gives you the concentration of the sample in the first well. Copy the formula down the column to calculate the concentrations of the rest of the samples.

The Excel sheet should look something like this, where the red text indicates user inputted data and the last column outputs the concentration of the samples:

• Remember to remove plate from the machine when you are done and clean it using bleach and UV.

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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 onto 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.

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Qiagen Multiplex Mix PCR for Multiple Band Amplification

Can be used for sexing, Rhodopsin amplification, etc.

For 2 μM Primer mix, can make master mix of:

• 20 μL Primer F (10 μM)
• 20 μL Primer R (10 μM)
• 60 μL dH₂O

PCR Mix:

Reagents	1x
Qiagen Multiplex Mix	5.0 μL
Q Solution	1.0 μL
BSA (10 mg/mL)	0.4 μL
Primer Mix (2 μM)	1.0 μL
dH2O	1.1 μL
DNA	1.5 μL
TOTAL	10.0 μL

PCR Cycle: MPLXST

• 95°C for 15 min
• For 12 cycles, -0.5°C per cycle (60→55°C):
  • 94°C for 30 sec
  • 60°C for 90 sec
  • 72°C for 60 sec
• For 32 cycles:
  • 94°C for 30 sec
  • 55°C for 90 sec
  • 72°C for 60 sec
• 60°C for 30 min
• 4°C forever

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HRM with Roche LightCycler 480

HRM Master Mix:

Reagents	1x
Primer Mix (2μM or 4μM)	0.50 μL
Roche MgCl2	1.75 μL
dH2O	2.25 μL
Roche MasterMix	5.00 μL
DNA (1ng+)	1.00 μL
TOTAL	10.5 μL

1. Sign up for a day and time to submit your plate on WebSeq under LightCycler 480 Signup.
2. Thaw reagents on ice, covered (Roche MasterMix is light sensitive!).
3. Make 2 μM primer mix by combining 1 μL of forward primer and 1 μL of reverse primer with 98 μL distilled water.
4. Make master mix and spin down.
5. Add 9.5 μL of the master mix to each well in a 384-well HRM plate.
6. Add 1 μL of DNA to each well and pipet mix.
7. Seal the plate with an HRM sticky lid and spin down.
8. Carry the plate on ice, covered, to the Core (CHS 36-125).
9. Ask personnel at the Core to run the HRM plate using the profile you need.

When the run is finished, the results will be posted on WebSeq under "Genotyping".

To view and analyze results:

1. Open LightCycler480 program. Make sure EXOR is running.
2. Log in and select "New Experiment".
3. Near the bottom, click the "Import" button, select your .ixo file, and run the relevant analysis.

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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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