Aug 02, 2024
alternative PCR protocol for gene COXI neo-caledonian freshwater micro-crustaceans
- Coline Royaux1,2,3,
- Nicolas Rabet1,2,3,
- Céline Bonillo1,2,3,
- Frédéric sson1,2,3
- 1Sorbonne Université;
- 2Muséum National d'Histoire Naturelle;
- 3UMR BOREA
- NC_THESIS_CR
Protocol Citation: Coline Royaux, Nicolas Rabet, Céline Bonillo, Frédéric sson 2024. alternative PCR protocol for gene COXI neo-caledonian freshwater micro-crustaceans . protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8q57v5b/v1
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: Working
We use this protocol and it's working
Created: August 02, 2024
Last Modified: August 02, 2024
Protocol Integer ID: 104483
Keywords: COI, PNDB, crustaceans, freshwater, alternative pcr protocol for gene coxi, cytochrome oxydase, pcr protocol, alternative pcr protocol, gene coxi, gene, streptocephalus with several primer
Abstract
This PCR protocol has been used to amplify the Cytochrome Oxydase I gene of neo-caledonian genera Boeckella, Lynceus, Latonopsis, Eulimnadia and Streptocephalus with several primers.
Prepare the mix for the PCR depending on the quantity of DNA samples you want to amplify
Note
The quantities per well and for 96 DNA samples (with a 4 volumes margin) are detailed in the sub-steps
Safety information
Be careful to take enough margin to avoid a lack of reagent and, if possible, do a positive and a negative control
For each well, mix 13.22 µL distilled water , 2 µL DNA buffer for Taq polymerase , 0.5 µL DNTP and 0.28 µL Taq polymerase . This mix is hereafter named "intermediary mix".
Note
Very few micropipettes are able to dose such a small amount so you'll have to make your mix for several wells at a time.
So, for 96 DNA samples with a 4 volume margin (100 volumes), mix 1322 µL distilled water , 200 µL DNA buffer for Taq polymerase , 50 µL DNTP and 28 µL Taq polymerase .
Add your primers to the mix, 0.5 µL selected H primer 10 picomolar (pM) and 0.5 µL selected L primer 10 picomolar (pM) for each well. This mix is hereafter named "final reagent mix".
Safety information
You may have to use different primers in your wells depending on the individual and the gene you want to amplify. If so, distribute the intermediary mix in as much tubes as primer pairs you want to use and add your primers in the proper quantity to make your final reagent mix. Don't forget to add margins in each mix for each different pair of primers you use.
Note
So, for 96 DNA samples with a 4 volume margin (100 volumes) and if you use the same primers for all 96 wells, add 50 µL selected H primer and 50 µL selected L primer
In each well, deposit 18 µL of the final reagent mix and 2 µL genomic DNA unless for the negative control.
For the positive control, use the DNA of the species on which the primer has been designed.
Close the PCR plate with a heated aluminium foil and centrifuge it enough to make sure the reagents are all in the bottom of the well.
To amplify targeted DNA sequences, place your plate in the thermocycler, set and launch the cycle as described in the following sub-steps
00:05:00 at 94 °C
5m
40 cycles of the following steps :
- 00:00:30 - 00:01:00 at 94 °C
- 00:00:30 - 00:01:00 at 45 °C - 52 °C
- 00:01:00 at 72 °C
4m
00:08:00 at 72 °C
8m
Infinite timing at 20 °C so the samples are well preserved and you have some time to pick it up
To know if your amplifications worked properly, you now have to proceed to a gel electrophoresis of your PCR products
To prepare the gel,
Safety information
This step is to be made under an extractor hood with gloves, a blouse and glasses as BET is highly mutagenic and carcinogenic
mix 0.5 mg agarose and 30 mL TAE buffer , heat the preparation in the microwave for a few moments until it is homogeneous (you can agitate the preparation). Wait until the preparation is cold enough to touch its container and add 0.5 µL BET .
Pour the preparation in a mold and let the gel polymerize with a bar to form wells to deposit your PCR products
Deposit the gel in the electrophoresis tank
Fill each well with 3 µL PCR product and 0.5 µL - 1.5 µL BG or any loading dye , don't forget to save a well to deposit 1.2 µL molecular weight marker
Start electrophoresis in TAE buffer for 00:15:00 at 200 Volts
15m
Look at your gel under Ultra Violet light to see the results