Determination of HIV RT activity was adapted from Willey et al. (21 (link)). In brief, collected culture supernatant (10 μl) was added to a cocktail containing poly (A) (GE Healthcare, Mickleton, NJ), oligo (dT) (USB, Cleveland, OH), MgCl2, and 32P dTTP (PerkinElmer, Boston, MA) and incubated for 20 h at 37°C. The cocktail (30 μl) was spotted onto DE81 paper, dried, and washed five times with 2 × saline-sodium citrate buffer and once with 95% ethanol. The filter paper was then air-dried. Radioactivity was counted in a liquid scintillation counter (PerkinElmer, Boston, PA).
Poly a
Manufactured by GE Healthcare
Poly A is a laboratory equipment used for the purification and isolation of polyadenylated RNA from biological samples. It functions by selectively binding to the poly(A) tail of mRNA molecules, allowing for their separation from other cellular RNAs.
Automatically generated - may contain errors
3 protocols using poly a
1
Quantification of HIV Reverse Transcriptase
2
Quantitative Analysis of Oligonucleotides in Tissues
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The ground tissue powder was suspended in 10 volumes/weight OTX Lysis Buffer, briefly needle-sonicated and centrifuged for 30 s at 14 000 rpm. Supernatants were collected, diluted at 1/750 in ultrapure water, and used for analysis. CL-qPCR was carried out following the protocol from Boos et al. (27 (link)), with minor modifications. For each tissue and each compound, a calibration curve was generated and run along with the samples. Mix composition for Chemical Ligation: 0.1 μl PS primer (10 μM stock), 0.1 μl BPS primer (10 μM primer), 1 μl Poly A (GE Healthcare #27411001, 1 mg/ml), 1 μl 10× Buffer with MgCl2 (Roche #12032902001) and 5.8 μl ultrapure water, mixed with 2 μl diluted lysate. Chemical ligation was run for 1 h at 33°C. qPCR was run on a LC480 device (Roche). A qPCR mix containing 0.15 μl FW primer (10 μM stock), 0.15 μl RV primer (10 μM stock), 0.13 μl BHQ primer (28.28 μM stock), 0.15 μl dNTPs mix (ThermoFisher #10297018), 1 μl 10× Buffer with MgCl2 (Roche #12032902001), 0.1 μl 10× FastTaq Polymerase (Roche #12032902001) and 6.32 μl ultrapure water was prepared and mixed with 2 μl of chemical ligation reaction mixture. qPCR program was as follows: 10 min, 95°C, 50 cycles (3 s/95°C – 30 s/55°C – 10 s/72°C). Concentrations in tissues were obtained through interpolation from the calibration curves and expressed in ng oligonucleotide/mg tissue for each mouse.
3
Quantification of Oligonucleotides in Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The ground tissue powder was suspended in 10 volumes/weight OTX Lysis Buffer, briefly needlesonicated and centrifuged for 30 s at 14'000 rpm. Supernatants were collected, diluted at 1/750 in ultrapure water, and used for analysis. CL-qPCR was carried out following the protocol from Boos et al. (28) , with minor modifications. For each tissue and each compound, a calibration curve was generated and run along with the samples. Mix composition for Chemical Ligation: 0.1 µl PS primer (10 µM stock), 0.1 µl BPS primer (10 µM primer), 1 µl Poly A (GE Healthcare #27411001, 1 mg/ml), 1 µl 10X Buffer with MgCl2 (Roche #12032902001) and 5.8 µl ultrapure water, mixed with 2 µl diluted lysate.
Chemical ligation was run for 1 h at 33°C. qPCR was run on a LC480 device (Roche). A qPCR mix containing 0.15 µl FW primer (10 µM stock), 0.15 µl RV primer (10 µM stock), 0.13 µl BHQ primer (28.28 µM stock), 0.15 µl dNTPs mix (ThermoFisher #10297018), 1 µl 10X Buffer with MgCl2 (Roche #12032902001), 0.1 µl 10X FastTaq Polymerase (Roche #12032902001) and 6.32 µl ultrapure water was prepared and mixed with 2 µl of chemical ligation reaction mixture. qPCR program was as follows: 10 min, 95°C, 50 cycles (3 s/95°C -30 s/55°C -10 s/72°C). Concentrations in tissues were obtained through interpolation from the calibration curves and expressed in ng oligonucleotide / mg tissue for each mouse.
Chemical ligation was run for 1 h at 33°C. qPCR was run on a LC480 device (Roche). A qPCR mix containing 0.15 µl FW primer (10 µM stock), 0.15 µl RV primer (10 µM stock), 0.13 µl BHQ primer (28.28 µM stock), 0.15 µl dNTPs mix (ThermoFisher #10297018), 1 µl 10X Buffer with MgCl2 (Roche #12032902001), 0.1 µl 10X FastTaq Polymerase (Roche #12032902001) and 6.32 µl ultrapure water was prepared and mixed with 2 µl of chemical ligation reaction mixture. qPCR program was as follows: 10 min, 95°C, 50 cycles (3 s/95°C -30 s/55°C -10 s/72°C). Concentrations in tissues were obtained through interpolation from the calibration curves and expressed in ng oligonucleotide / mg tissue for each mouse.
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