Individual sequences for RBD variants were ordered as complementary forward and reverse primers (Integrated DNA Technologies) in 96-well plates A single round of annealing and extension was used to produce double-stranded DNA with 14-bp of homology at 5’ and 3’ ends to the pYD1-RBD entry vector, followed by Gibson Assembly with EcoRI digested vector. Plasmids were transformed into EBY100 prepared with the Frozen-EZ Yeast Transformation Kit II (Zymo) and plated on SD-UT agar. Individual colonies were picked and grown in SD-UT liquid medium overnight at 30°C, then diluted to OD600 = 0.5 in SG-UT medium and grown for 40-48 hours at 23°C. Cells were stained with biotinylated ACE2 or purified antibody as described above. Flow cytometry analysis was performed on the BD Fortessa cytometer.
Complementary forward and reverse primers
Manufactured by Integrated DNA Technologies
Complementary forward and reverse primers are nucleic acid sequences used in various molecular biology techniques, such as polymerase chain reaction (PCR), to amplify specific regions of DNA. These primers are designed to be complementary to the target DNA sequence, allowing the DNA polymerase to initiate DNA synthesis and generate multiple copies of the desired DNA fragment.
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Lab products found in correlation
2 protocols using complementary forward and reverse primers
1
RBD Variant Display and Screening
2
RBD Variant Expression and Screening
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Individual sequences for RBD variants were ordered as complementary forward and reverse primers (Integrated DNA Technologies) in 96-well plates A single round of annealing and extension was used to produce double-stranded DNA with 14-bp of homology at 5' and 3' ends to the pYD1-RBD entry vector, followed by Gibson Assembly with EcoRI digested vector. Plasmids were transformed into EBY100 prepared with the Frozen-EZ Yeast Transformation Kit II (Zymo) and plated on SD-UT agar. Individual colonies were picked and grown in SD-UT liquid medium overnight at 30°C, then diluted to OD600 = 0.5 in SG-UT medium and grown for 40-48 hours at 23°C. Cells were stained with biotinylated ACE2 or purified antibody as described above. Flow cytometry analysis was performed on the BD Fortessa cytometer.
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