The complete HPV16 genome was amplified in a 25 μL solution with 2.5 μL (15.5 ng) of extracted nucleic acid from passage 2, 0.2 μmol/L of each primer (HPV16 7465F, 5′-ATGCTTTTTGGCACAAAATGTG, HPV16 7464R 5′-GCAACCGAATTCGGTTGAAG), 200 μmol/L of each dNTP, 1 × PrimeStar GXL Buffer, and 0.625 U PrimeStar GXL DNA Polymerase (TaKaRa Bio, Shiga, Japan). The PCR was performed at 98 °C for 10 s, 60 °C for 10 s, and 68 °C for 8 min for 45 cycles. Amplified DNA was separated by electrophoresis in a 1.0% agarose gel (agarose Type I-A, A0169, Sigma-Aldrich, Stockholm, Sweden) with 1 × Gel RedTM Nucleic Acid Gel Stain (VWR International, Lund, Sweden) in 0.5 × TBE buffer [23 ] and visualized by UV light.
Agarose type 1 a
Manufactured by Merck Group
Sourced in United States
Agarose Type I-A is a purified polysaccharide extracted from red seaweed. It is commonly used as a gelling agent in various laboratory applications, such as gel electrophoresis and cell culture. Agarose Type I-A exhibits a high gelling strength and is suitable for a variety of analytical and preparative techniques.
Automatically generated - may contain errors
8 protocols using agarose type 1 a
1
Amplification of HPV16 Genome
2
MMP9 Enzyme Cleavage Assay
3
Ultra-Low Gelling Agarose Resuspension
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An ultra-low gelling temperature (ULGT) agarose (Agarose Type IX-A, Sigma-Aldrich, St. Louis, MO, USA) that gels at temperature <17°C was used as a resuspending medium. A standard agarose (Agarose Type I-A, Sigma-Aldrich) that gels at <36°C was used as a re-embedding medium. Each agarose material was melted in boiling water at 3% (w/v). In order to preserve gelation quality of the agarose solutions, they were kept at 4°C with the cap fully tightened. When ready to be used, they were re-melted using a microwave oven. The re-melted ULGT agarose solution was then kept at room temperature while the re-melted standard agarose solution was kept in the oven set at 60°C to prevent premature solidification prior to use.
4
Agarose Pre-embedding of Cell Pellets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two types of agarose solutions were used in order to pre-embed the cell pellets. Three percent (w/v) ultra-low gelling temperature (ULGT) agarose (Agarose Type IX-A, Sigma-Aldrich, St. Louis, MO, USA) that has a gelling temperature of < 17°C was used as a resuspending medium. Three percent standard agarose (Agarose Type I-A, Sigma-Aldrich) with a gelling temperature of 34-38°C was used as a re-embedding medium. In order to preserve the gelation quality of the agarose solutions, they were kept at 4°C with the cap fully tightened. When ready to be used, they were re-melted using a microwave oven. The re-melted ULGT agarose solution was then kept at room temperature while the re-melted standard agarose solution was kept in an oven set at 60°C to prevent premature solidification prior to use.
5
FADS2 and FABP4 Gene Amplification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The primers presented in Table 1 were used to amplify fragments of the FADS2 and FABP4 genes. PCR reactions (20 µL) were performed in an AG22331 thermocycler (Eppendorf, Hamburg, Germany). The mixture consisted of 1x PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTP, 0.2 µM of each primer, 1.2 U Taq DNA polymerase I (Thermo Scientific, Vilnius, Lithuania), and 150–200 ng DNA. Thermal program for each gene included the following steps: initial denaturation at 94 °C for 2 min, 35 cycles of proper denaturation at 94 °C, primer annealing (Table 1 ), elongation at 72 °C for 30 s, and final elongation at 72 °C for 5 min. Fragments of the amplified genes were cut with restriction enzymes for 4 h (Table 1 ) and separated on a 2% agarose gel (Agarose Type I-A, Sigma Aldrich, St. Louis, MO, USA) in 1x TBE buffer; visualization was performed using BOX Chemi XR5 (Syngene, Cambridge, England).
6
Acute Slice Preparation of Mouse Vomeronasal Organ
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Acute slices of mouse VNO were prepared as previously described (Shimazaki et al., 2006 (link); Dibattista et al., 2008 (link); Wong et al., 2018 (link)). In brief, the VNO was removed and transferred to ice-cold artificial CSF (ACSF) containing the following: 120 mm NaCl, 20 mm NaHCO3, 3 mm KCl, 2 mm CaCl2, 1 mm MgSO4, 10 mm HEPES, and 10 mm glucose, pH 7.4. The capsule and all cartilaginous tissues were carefully removed and the two halves of the VNO were isolated from the vomer bone. Each half of the VNO was then separately treated. The VNO was embedded in 3% Type I-A agarose (Sigma) prepared in ACSF once the agar had cooled to 38°C. Upon solidification, the agar block was fixed in a glass Petri dish and sliced with a vibratome (Vibratome 1000 Plus Sectioning System) at 200 to 250 μm thickness in oxygenated ACSF solution. Slices were then left to recover for >30 min in chilled and oxygenated ACSF before electrophysiological experiments were initiated.
7
Fabricating Agarose Microwells for Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For a video version of the protocol, follow this link: https://figshare.com/s/0d3706f760854a44f603 .
An expanded version of the protocol, along with hints and tips, is available in the supplementary information (Suppl. Protocol 1 https://figshare.com/s/1795d13ce724ebfa0ee9 .
Silicone release spray (Bond It) was sprayed on the mold maker and left to dry (30 s) to facilitate separation from the agarose gel. Type IA agarose (A0169, Sigma-Aldrich, St. Louis, MO) was dissolved by microwaving in deionized water to make a 2% solution. The hot agarose solution was kept at 50–70 °C in a water bath, and 2 mL was dispensed in a prewarmed (37–50 °C) stainless steel histology base mold (Simport M474-4, 30 × 24 × 5 mm). The mold maker was placed on top of the warm agarose solution, and the base mold gently pressed and tapped to remove any potential air bubbles trapped underneath the mold maker. The agarose solution was left to gel at room temperature (2 min, 21 °C), and subsequently the mold was transferred to a laboratory freezer and placed on a level surface (1 min, −18 °C). The mold maker was removed, leaving an agarose mold of 66 wells.
An expanded version of the protocol, along with hints and tips, is available in the supplementary information (
Silicone release spray (Bond It) was sprayed on the mold maker and left to dry (30 s) to facilitate separation from the agarose gel. Type IA agarose (A0169, Sigma-Aldrich, St. Louis, MO) was dissolved by microwaving in deionized water to make a 2% solution. The hot agarose solution was kept at 50–70 °C in a water bath, and 2 mL was dispensed in a prewarmed (37–50 °C) stainless steel histology base mold (Simport M474-4, 30 × 24 × 5 mm). The mold maker was placed on top of the warm agarose solution, and the base mold gently pressed and tapped to remove any potential air bubbles trapped underneath the mold maker. The agarose solution was left to gel at room temperature (2 min, 21 °C), and subsequently the mold was transferred to a laboratory freezer and placed on a level surface (1 min, −18 °C). The mold maker was removed, leaving an agarose mold of 66 wells.
8
Preparation of Human Nasal Epithelium Slices
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Acute slices of human nasal epithelium used for electrophysiological experiments were prepared following a similar protocol to the one used for mouse olfactory and vomeronasal epithelium.15 (link),62 (link),77 (link),78 (link),79 (link),80 (link),81 (link) Within about 30 min from the biopsy, the human nasal epithelium was embedded in 3% Type I-A agarose (Sigma) prepared in ACSF once the agar had cooled to 38°C. Upon solidification, the agar block was fixed in a glass Petri dish and sliced with a vibratome (Vibratome 1000 Plus, Sectioning System) at 200 to 250 μm thickness in oxygenated ACSF solution. Slices were then left to recover for >30 min in chilled and oxygenated ACSF before electrophysiological experiments were initiated.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!