3' UTR RACE Protocol with Sanger Sequencing

For the 3′ UTR RACE, reverse transcription was realized with an oligo-dT adapter primer: (GCTGTCAACGATACGCTACGTAACGGCATGACAGTGTTTTTTTTTTTTTTTTTTTTTTTT) as described (9 (link)). PCRs were performed in a final volume of 15 μl with 1 μl of RT product, 3 μl of reverse primer at 20 μM and 1 μl of primer at 20 μM, 7.5 μl 2× PCR MasterMix (ThermoScientific). The PCRcycling conditions were 94°C for 5 min, followed by 5 cycles at 94°C for 20 s and 72°C for 50 s, then 5 cycles at 94°C for 20 s and 70°C for 30 s and 72°C for 20 s and 25 cycles at 94°C for 20 s and 68°C for 20 s and 72°C for 30 s, followed by a final extension at 72°C for 7 min. The nested PCRs were realized in a final volume of 15 μl with 1 μl of primary PCR product, 1 μl of reverse and/or forward primers 20 μM, 7.5 μl 2× PCR MasterMix (Thermo Scientific).The PCR cycling conditions were 94°C for 5 min, followed by 35 cycles at 94°C for 20 s and 60°C for 20 s and 72°C for 30 s, followed by a final extension at 72°C for 7 min. Nested PCR products were purified using a NucleoSpin Gel and PCR Clean-Up column (Macherey-Nagel).Fragments were cloned into TOPO-TA vector with a TOPO-TA Cloning kit (LifeTechnologies).Cleavage sites were determined by Sanger sequencing of at least 10 Individual clones.

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Dion C., Roche S., Laberthonnière C., Broucqsault N., Mariot V., Xue S., Gurzau A.D., Nowak A., Gordon C.T., Gaillard M.C., El-Yazidi C., Thomas M., Schlupp-Robaglia A., Missirian C., Malan V., Ratbi L., Sefiani A., Wollnik B., Binetruy B., Salort Campana E., Attarian S., Bernard R., Nguyen K., Amiel J., Dumonceaux J., Murphy J.M., Déjardin J., Blewitt M.E., Reversade B., Robin J.D, & Magdinier F. (2019). SMCHD1 is involved in de novo methylation of the DUX4-encoding D4Z4 macrosatellite. Nucleic Acids Research, 47(6), 2822-2839.

Publication 2019

2× PCR MasterMix NucleoSpin Gel and PCR Clean-Up column TOPO-TA Cloning kit

3 utr Cleavage Clones Nested pcr Oligo dt Primers Reverse transcription Topo Vector

Corresponding Organization : Aix-Marseille Université

Other organizations : University College London, Great Ormond Street Hospital, Agency for Science, Technology and Research, Institute of Molecular and Cell Biology, Institute of Medical Biology, University of Melbourne, Walter and Eliza Hall Institute of Medical Research, Université de Montpellier, Centre National de la Recherche Scientifique, Institut de Génétique Humaine, Délégation Paris 5, Sorbonne Paris Cité, Université Paris Cité, Institut des Maladies Génétiques Imagine, Hôpital de la Timone, Hôpital Necker-Enfants Malades, Assistance Publique – Hôpitaux de Paris, Mohammed V University, National University of Singapore, Academic Medical Center

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Variable analysis

independent variables

Reverse transcription with oligo-dT adapter primer
PCR conditions (number of cycles, temperatures, durations)
Nested PCR conditions (number of cycles, temperatures, durations)

dependent variables

3' UTR sequences

control variables

Final volume of PCR reactions (15 μl)
Concentration of reverse and forward primers (20 μM)
PCR MasterMix (Thermo Scientific)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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