Mar 30, 2023
PCR on DNA samples (Chlamydomonas spp.) to test annealing temperature for the primers
This protocol is a draft, published without a DOI.
- 1University of Konstanz
Protocol Citation: Richard W Lambrecht, Manuela Spagnuolo 2023. PCR on DNA samples (Chlamydomonas spp.) to test annealing temperature for the primers. protocols.io https://protocols.io/view/pcr-on-dna-samples-chlamydomonas-spp-to-test-annea-cr5fv83n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: March 30, 2023
Last Modified: March 30, 2023
Protocol Integer ID: 79751
Keywords: PCR, annealing temperature test
Abstract
Protocol developed to test the annealing temperature of primers (ITS, rbcL and 18S regions).
Initial observations
Initial observations
Make sure you had appropriate training/introduction to the laboratories and machines (From Dr. Epp)
Book the laboratory benches and machines in advance
Respect the rules for each laboratory and the transit between the laboratories
Pre-PCR Lab
Pre-PCR Lab
Take Primers, ultrapure water, MasterMix, and DNA out of the freezer and defrost completely
Primer dilution (Primer stock concentration 100 µM) to a 10 µM Primer
Take defrosted primer and mix by flicking the tube (do not vortex!!)
Aliquot 45 µl of ultra-pure water in a clean 1.5 ml Eppendorf tube and add 5 µL of the 100 µM primer to dilute it to a 10 µM concentration
Prepare the gradient reaction volume (for 14 reactions – 12 reactions + 2 extra)
* Number of reactions = number of wells per primer combination X 15 µL per well.
**Primer combinations list is on the last page of this protocol.
In 1.5 mL tubes:
- Water: 92.4 µL
- Hot Start Master Mix (TaqPolymerase): 105 µL: make sure the MasterMix is defrosted completely and vortex 20 seconds before adding to ensure proper mixing
- Forward Primer: 4.2 µL: flick before adding
- Reverse Primer: 4.2 µL: flick before adding
- Template/DNA: 4.2 µL: flick the tube before adding
Flick all the 1.5 ml tubes with the mixed PCR reagents and spin them down using the quick-spin before adding them to the PCR plate
Seal plates using transparent sealing foil. Make sure that the plates are sealed correctly; otherwise, the samples will evaporate in the cyclers.
Post-PCR Lab
Post-PCR Lab
Put the plates in the cyclers in the post-PCR lab
Settings for the cyclers:
- Jena Analytics cycler - protocol name: richard (Temperature range: 46ºC to 56ºC)
- PeqLab2 – protocol name: 45-GRARICH (Temperature range: 58ºC to 68ºC)
a) 95ºC for 15 min (Hot Start for the Master Mix)
b) 35 cycles
i) [ 94ºC for 30 sec
ii) Temperature gradient for 30 sec (Chosen temperature gradient)
iii) 72ºC for 1:30 min (*for Taq DNA calculate 1 min per Kb of target DNA)]
c) 72ºC for 10 min.
Prepare the gel for the electrophoresis
[Fume hood] Prepare the gel tray by sealing the openings with lab tape. Make sure the tape is sticking properly and seals the openings, otherwise, your gel will leak
Put the combs with the number of wells you want to have in the tray (decide the amount of wells based on the amount of wells + at least 1 leader per row of wells)
For a 1.5% small gel:
i) Weigh in 0.75 g of agar (labeled with LB) and put it into a 100 ml Erlenmeyer flask
ii) Add around 60 mL of 1x TAE buffer (left side of the electrophoresis bench)
For a 1.5% big gel:
i) Weigh in 1.5 g of agar (labeled with LB) and put it into a 200 ml Erlenmeyer flask
ii) Add around 110 mL of 1x TAE buffer (left side of the electrophoresis bench)
Block the opening of the Erlenmeyer flaks with a small piece of paper. Make sure you are not completely blocking the opening. There should be still some air exchange possible. This will prevent you gel from boiling over in the microwave
Put the Erlenmeyer flask in the microwave until it is completely clear. Take the flask out every 30 seconds- 1 min to twirl it. This will ensure proper mixing
[Fume hood] Let the gel cool until you can touch the bottom of the flask for 5 seconds with gloves on otherwise the tape will not stick to the tray anymore. Twirl the flask every 2 minutes to ensure proper mixing
Add 5 µl of a 10 000X SYBR save (shelf on top of the microwave) to the small gel, for the big gel it is 10 µl to get a 1X concentration of SYBR safe
Twirl the flask to mix the SYBRsafe properly and immediately pour the gel into the tray to avoid clumping
Let it cool and harden (approx. 10 min) check with the broad end of a pipette tip if the gel is solid
Carefully remove the tape from the tray and place the tray with the gel in the electrophoresis chamber. Make sure the TAE buffer in the chamber completely covers the gel. The TAE should be 5 mm above the gel. If the TAE in the chamber is not enough pour some more from the bottle
Remove the combs carefully to not damage the wells of the cell. If you rip one of the wells, do not use it for loading anymore. Make sure that the TAE filled ever well of the gel
Stain the sample to load the gel for the electrophoresis
a) Aliquot approx. 1 µl of a 6X loading dye (green) to six small wells of a PCR loading tray (next to the sink) using a 20 µl pipette set to 8 µl
b) Add 5 µl of sample to each well. Mix by pipetting up and down 3 times and load it directly to the gel. Change the tip between every sample to avoid contamination
Load 3.5 µL of the leader (Gene ruler SM#0333) in the middle of each row of the gel for the electrophoresis, or in between samples to separate blocks. Change tips in between loading each ladder well
Running the gel
Close the electrophoresis chamber with the according lid. Make sure the connections are correct (red to red; black to black)
Mark on the tape on the lid that you ran a gel (TAE buffer in the chamber needs to be exchanged after 10 runs)
Connect the cables to the power supply
Chose the program with the voltage you want to run your gel (100 V should be program 1)
Time: Small gel (~30 min), big gel (~40 min). Make sure you check your gel in between. You do not want your samples to run it off the gel.
You can stop after the gel ran at least 2 cm from the well by switching the electrical device off
Gel analysis
Start the analysis computer
Open the “intas” software
Take the gel tray out of the electrophoresis chamber, use paper towels to dry the bottom of the tray and close the chamber after you are done
Place the gel with the tray on the table of the analysis device
Switch on the normal light in the device (NOT the UV light!!!!) and press “live” on the software. This allows you to see a live image to position your gel correctly
Change the position of the gel so that you can get an even image of your gel
You can focus and zoom in on your gel by turning the objectives of the camera on top of the analysis device.
When your gel is positioned correctly, close the door of the analysis device and switch on the UV light
Change exposure time until you get a clear image of your gel and your bands/ the ladder bands can be seen clearly
Press “freeze” on the software and switch off the UV light. This is protecting the UV light.
Save and print the picture of your gel
Take the gel tray of the device and discard the gel into the gel waste (white bin with blue lid)
Clean the tray, the Erlenmeyer flask and the plastic PCR sample tray with hot water