Unless stated otherwise, all chemicals were from Sigma-Aldrich (St. Louis, MO). Oligonucleotides were purchased from MWG Biotech AG (Ebersberg, Germany). Pfu Turbo DNA polymerase was purchased from Agilent Technologies. All other enzymes were from Fermentas. The plasmid pGEM-1 and the TNT® SP6 Transcription/Translation System were from Promega. [35S]Met was from PerkinElmer.
Pgem 1
Manufactured by Promega
Sourced in United States
pGEM®-1 is a small, high-copy-number plasmid vector used for cloning and sequencing applications. It contains the lac promoter and lac Z alpha gene for blue/white colony screening, as well as multiple cloning sites for insertion of DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
35s met, by PerkinElmer (4 mentions)
Tnt sp6 transcription translation system, by Promega (3 mentions)
Pfu turbo dna polymerase, by Agilent Technologies (3 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
N1660, by Merck Group (1 mentions)
Deoxynucleotide, by Promega (1 mentions)
Tnt quick transcription translation system, by Promega (1 mentions)
Oligonucleotides, by Eurofins (1 mentions)
Pgem2, by Promega (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Dig rna labeling kit sp6 t7, by Roche (1 mentions)
6 protocols using pgem 1
1
Recombinant Protein Expression Protocol
2
Immunoblotting and Aβ Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used for immunoblotting: NT-1 recognizing the PS1-NTF [61] (link), α-loop (Chemicon) raised against PS1-CTF, N1660 (Sigma) raised against the C-terminal of nicastrin, 3891 (ProSci Inc.) raised against Pen-2 and α-GAPDH (Acris GmbH) recognizing GAPDH. For the quantification of secreted Aβ38, 40 and 42 with Meso Scale Discovery (MSD) technology, C-terminal specific antibodies (Aβ 1-x) were used and detection was performed by SULFO-TAG™ 6E10 antibody. For quantification of sectreted Aβ40 and Aβ43 using Aβ40 Wako II ELISA kit (Wako Chemicals GmbH) and FL 1-43 ELISA kit (Immuno-biological Laboratories), respectively, the capture antibody was BNT77 for Aβ40 and Aβ38-43 for Aβ43. Detection antibodies were BA27 (Aβ40) and 82E1 (Aβ43), respectively. Unless otherwise stated, all chemicals were from Sigma–Aldrich. Plasmid pGEM1, TNT® Quick transcription/translation system, and deoxynucleotides were purchased from Promega and 35S-Met from PerkinElmer. All enzymes were obtained from Fermentas except Phusion DNA polymerase that was from Finnzymes. Oligonucleotides were from Eurofins MWG Operon.
3
Synthetic tRNA Preparation and Labeling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The template plasmid for human tRNAiMet-CAT-2 encoding T7 promoter, hammerhead ribozyme, and nucleotides 9–72 of the tRNA followed by CCA was generated by overlap extension PCR and cloned in pGEM-1 (Promega). The templates for human tRNAAsp-GTC-2 and the variant tRNAAsp-GTC-A9G were generated by gene synthesis (BIOMATIK), and encoded T7 promoter, hammerhead ribozyme, nucleotides 9–72 of the tRNA followed by CCA, and a 3′ HDV ribozyme. The tRNAiMet-CAT-2 and the two tRNAAsp-GTC templates were cleaved with BstNI and HindIII, respectively, and the tRNA fragments were produced as run-off transcripts guided by T7 RNA polymerase. The in vitro transcribed tRNA fragments were 5′-end labelled and ligated by splint-guided ligation to a synthetic RNA corresponding to the first eight nucleotides of the respective tRNA. In vitro transcription, 32P labeling at position 9, purification, and splint-guided ligation were carried out as previously described (10 (link)).
4
Recombinant Protein Expression Protocol
5
In situ Hybridization of Mouse Placental Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse Tpbpa and Pl-1 cDNA fragments cloned in pGEM1 and pGEM2 (Promega,
Madison, WI, USA), respectively, were kindly provided by Dr. Janet Rossant (Samuel Lunenfeld Research
Institute, Mount Sinai Hospital, Toronto, Canada). A 257-bp fragment of Plpa was amplified by
polymerase chain reaction (PCR) using cDNA from a placenta at E12.5 as the template with the following
primers: Plpa-F, 5’-gatcccagcccttcacacat-3’, and Plpa-R,
5’-ggcaatccagttatcccaga-3’. The PCR products were ligated into pGEM-T Easy Vector (Promega), cloned and
sequenced using an ABI DNA sequencer (Applied Biosystems, Foster City, CA, USA). The plasmid DNAs were
linearized, and sense and antisense digoxigenin (DIG)-labeled riboprobes were generated using a DIG RNA
Labeling Kit (Sp6/T7) (Roche, Mannheim, Germany).
Cryosections (20 µm thick) were placed on slides, and ISH was performed as described previously [13 (link)]. After hybridization with DIG-labeled probes, the slides were
counterstained with eosin, dehydrated, cleared in xylene and mounted in Canada balsam.
Madison, WI, USA), respectively, were kindly provided by Dr. Janet Rossant (Samuel Lunenfeld Research
Institute, Mount Sinai Hospital, Toronto, Canada). A 257-bp fragment of Plpa was amplified by
polymerase chain reaction (PCR) using cDNA from a placenta at E12.5 as the template with the following
primers: Plpa-F, 5’-gatcccagcccttcacacat-3’, and Plpa-R,
5’-ggcaatccagttatcccaga-3’. The PCR products were ligated into pGEM-T Easy Vector (Promega), cloned and
sequenced using an ABI DNA sequencer (Applied Biosystems, Foster City, CA, USA). The plasmid DNAs were
linearized, and sense and antisense digoxigenin (DIG)-labeled riboprobes were generated using a DIG RNA
Labeling Kit (Sp6/T7) (Roche, Mannheim, Germany).
Cryosections (20 µm thick) were placed on slides, and ISH was performed as described previously [13 (link)]. After hybridization with DIG-labeled probes, the slides were
counterstained with eosin, dehydrated, cleared in xylene and mounted in Canada balsam.
6
Recombinant Protein Expression Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Unless stated otherwise, all chemicals were from Sigma-Aldrich (St Louis, MO, USA). Oligonucleotides were purchased from MWG Biotech AG (Ebersberg, Germany). Pfu Turbo DNA polymerase was purchased from Agilent Technologies. All other enzymes were from Fermentas. The plasmid pGEM-1 and the TNT SP6 Transcription/Translation System were from Promega. [35S]Met was from PerkinElmer.
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!