...using the agarose cloning method Test your agarose temperature. Cloning cells using an agarose overlay ...to pour the agarose onto the recipient plate at just the right temperature. If the agarose solution is...the plate. We test the (capped) 1:1 agarose/DMEM solution against a part of the body very sensitive to...gene of interest. Advantages of the agarose cloning method The agarose technique should be useful to scientists...containing a low percentage of agarose, which is then overlaid with the firmer 2% agarose gel solution discussed...aerated with a thin layer of molecular biology-grade agarose containing physiological concentrations of salts...visible to the naked eye) with a marker prior to agarose addition, we found it was possible to rapidly punch...

...for generating sybodies against membrane proteins Developing antibodies against proteins embedded in membranes... challenging when trying to develop antibodies against unstable targets like membrane proteins that may...plasmids to generate sybodies, synthetic nanobodies against the spike protein (Walter et al., 2020). No llamas...selection of synthetic nanobodies, called sybodies, against membrane protein targets in as little as 3 weeks...Outline of the process for selection of sybodies against membrane proteins. Image from Zimmermann et al....Seeger MA (2020) Generation of synthetic nanobodies against delicate proteins. Nature Protocols 15:1707–1741...

...synthetic biology, biosecurity, and the entire 2018 saga of “what’s happening in genetics this time?” She...

...efforts. So, how effective are the current vaccines against the variants? Alejandro Balazs, a Group Leader...study. Figure 1: Neutralization levels against spike pseudovirus decreases depending on the variant...studies on the effectiveness of current vaccines against SARS-CoV-2 variants, this study was the first to...

...of LB-agar, we’ll need: (37 g pre-mixed LB-agar powder/L) x (0.220 L) = 8.14 g pre-mixed LB-agar powder... Protocols Pouring LB Agar Plates Pouring LB Agar Plates...peptone from casein 10.0 g sodium chloride 12.0 g agar-agar 1 L Sterile H 2 O Sterile plates of your desired...Procedure Measure 37g of pre-mixed LB-agar powder per L of molten agar you’d like to make. The precise mass...autoclaving. We make 400 mL of agar in 1 L bottles and 200 mL of agar in 500 mL bottles. The extra empty volume...Cooled agar should be warm to the touch; as a rule of thumb, if you cannot take the molten agar out of.... Swirl the agar bottle to ensure even distribution of the antibiotic throughout the agar. Open one plate...

...Procedure Pouring a Standard 1% Agarose Gel: Measure 1 g of agarose. Pro-Tip Agarose gels are commonly used in...mass of agarose in a given volume to make gels of other agarose concentrations (e.g., 2 g of agarose in 100... Protocols Agarose Gel Electrophoresis Agarose Gel Electrophoresis You may also ...adjust the agarose percentage of the gel to get better separation. A higher percentage agarose gel will ...Standard protocol for performing agarose gel electrophoresis, including tips to improve resolution and...Restriction Digest of Plasmid DNA Purifying DNA from an Agarose Gel DNA Ligation Introduction Gel electrophoresis... to move the negatively charged DNA through an agarose gel matrix toward a positive electrode. Shorter...

...Bacteria on LB Agar Plate Over-Agar Antibiotic Plating You may also like... Making LB Agar Plates Bacterial...plating antibiotic over-agar for the selection of transformed E. coli . Over-agar spreading of antibiotic...containing solidified LB-agar, pipette 150 µL of carbenicillin on top of the agar and gently spread over...plated over-agar for selection of transformed E. coli . Sample Data Selection of E.coli on LB-agar using different...prepare separate batches of antibiotic-containing agar. This protocol will focus specifically on selection...video below to learn how to spread antibiotic over-agar. Equipment Pipette tips for both pipetting and spreading...flame source) Incubator Reagents 6 cm diameter LB/agar plates without antibiotic High concentration (100...

...Protocols Purifying DNA from an Agarose Gel Purifying DNA from an Agarose Gel You may also like... Restriction...Protocol for how to purify DNA from an agarose gel, including tips to help improve the resolution and...Restriction Digest of Plasmid DNA Agarose Gel Electrophoresis DNA Ligation Background Information Gel purification...based on size. The procedure starts with standard agarose gel electrophoresis, which separates DNA by their...electrophoresis, you can cut DNA bands out of the agarose gel and purify the DNA samples. This is a commonly...cloning. Protocol: Gel Purification Follow the Agarose Gel Electrophoresis Protocol with the following...Gel purification is most efficient with lower % agarose gels, so you will want to stay in the 0.7-0.8% ...

...GCC, GCA, GCG Arginine Arg R CGU, CGC, CGA, CGG, AGA, AGG Asparagine Asn N AAU, AAC Aspartic acid Asp ...allow for the propagation of the plasmid within bacteria, while allowing for selection against any bacteria...bacteria with plasmids, thus selecting against the propagation of these plasmids through cell division...Introduction Types of Plasmids E. coli strains for propagating plasmids Antibiotics commonly used for plasmid...technology, which enables the preparation of more managable (i.e., smaller) DNA fragments. In 1952, Joshua...more. Regardless of type, plasmids are generally propagated, selected for, and the integrity verified prior...to use in an experiment. E. coli strains for propagating plasmids E. coli are gram-negative, rod shaped...

... aak1 TAL3208 & TAL3209 TCTTTGACTCCCGGAGAGagctggtgggctccgggCCAGGTTCTGGAGGGGGA abcg2a TAL3400 & TAL3401...ahcyl1 TAL3018 & TAL3019 TGACAGACTGTGGCGAGGatgccaagctggaggcGACGAAACAGGCCAGCAA amh TAL3210 & TAL3211 TCCAGGCAAGATTTGGGCtgatgctgatgatgacCGTGGCGATTGGGTCGTA...TGATCTGGATCCTAACAAggatgacttgcccctgtGTGCGAACACCAACCATA arf6 TAL3212 & TAL3213 TGGGGAAGATGCTTTCCAagatcttcggcaacaaGGAGATGAGAATATTGA arnt1 TAL3214 & TAL3215 TCCCGTCTCTGGCCATGAcctcctctaatggatcTCATTCCAACGGTGTGCA...apoa4-like TAL3023 & TAL3024 TGAAGGTTCTTGTGGTGCtcacacttgctgtggtTACAGGTAAGAAACTAAA apoeb TAL3402 & TAL3403 ...TAL3403 TGCCAGGCTCGTAGCCTGttccaggctgatgccccTCAGCCCAGATGGGAGGA arap3a TAL3026 & TAL3027 TGGCAACAATCGGTCCGTgctcagatgcagaggaCCTGTTGGCTTCCATCCA...ARSA TAL 3002 & TAL 3003 TCAAGAGGCGGTCTCCCTctcaacgagaccaccaTAGCCGAGGTGCTGAAGA asun TAL3404 & TAL3405 TAACTCAAGCCAACTCCAggaaacatcaggagtttGCAGGAAGATTGAGTTCA... TAACTCAAGCCAACTCCAggaaacatcaggagtttGCAGGAAGATTGAGTTCA atf6 TAL3406 & TAL3407 TCCAACCAGCAAAAAGACccatccaggtgtgccccaAGGTTTCCATTCAGCCTA...

...victoria GGTGGTGCAGATGAACTTCA 47513 cut S. pyogenes 23792628 Joung EGFP A. victoria TGAAGAAGATGGTGCGCTC 58255...sapiens GATACTGGAGAAGCAGGGGC 68466 cut S. pyogenes 26145478 Zhang PAX6 H. sapiens GTAGATTTTGTATGCACTGC 68465...Johnson UCA1 H. sapiens gRNA1:GGTTTCCTTTTAGATGACGG; gRNA2:TCTGAAAAGAGAGTCAGCGA 72628 cut S. pyogenes 26493208...pyogenes 25739462 Jiang ade6-L469 S. pombe TCTATTGTTCAGATGCCTTG 52227 cut S. pyogenes 25352017 Zaratiegui...Zaratiegui ade6-M210 S. pombe TCTATTGTTCAGATGCTTCG 52226 cut S. pyogenes 25352017 Zaratiegui ade6+ S. pombe TCTATTGTTCAGATGCCTCG... TCTATTGTTCAGATGCCTCG 52225 cut S. pyogenes 25352017 Zaratiegui Alk and Eml M. musculus 64071 cut S. pyogenes...pyogenes 26472758 Sabatini ANT1 S. lycopersicum TGTCGTTTATAATTTGTAGA 70019 cut S. pyogenes 26541286 Voytas APC...

...OPEN OPEN aGACTCCGTCccgtcgtGCTGAGGGCc hnf1a OZ521 and OZ522 OPEN OPEN aACCTTCCCCagagaGAGGTAGTGg VDAC2 OZ523...OPEN OPEN gCCCCTCAGCccagatgGGAGGAGATg dpf2 OZ531 and OZ532 OPEN OPEN cTGCTACTTCtggatGGAGAAACGa pitx2 OZ533... CODA tGTCTTCATCTCCTTCAGATGCTGTCc fam82a2 OZ573 and OZ574 CODA CODA gCACCGCTGCAGTAAGAGCTGAGGAAt morc3b...CODA CODA aGTCCGCCTCCACCCGAGGTAGAG zgc:111821 OZ597 and OZ598 CODA CODA tGGCAGCAGCTTTTCGAAGAATAGg zgc:65890...half-site CACNA1F OZ507 and OZ508 OPEN OPEN gCGTGACCTCgctgatAAGAAGAGg xylt1 OZ509 and OZ510 OPEN OPEN cACCCTCTACaggacaGTAGCGGGTg...gACAGCCGTCtgcatctGGAGGTGCCc pitrm1 OZ515 and OZ516 OPEN OPEN cACCTGCAGCagattGAAGAAGAGg cftr OZ517 and OZ518 OPEN OPEN gTGCAACCGCagctttTAGGACGGAt...Encephalopsin (Opn3) OZ541 and OZ542 OPEN OPEN aTCCATCCCAaacacatGTGGCAGAAt NFATc1 OZ543 and OZ544 OPEN OPEN aCCTCGCCTCagtgtGACGGAGGAc...

...Prefix: GAATTC GCGGCCGC T TCTAGA G Alternate Prefix: GAATTC GCGGCCGC T TCTAGA TG (contains start) Prefix...CTGCAG Suffix Enzymes: SpeI , NotI , PstI Scar: TACTAGAG or TACTAG Features: 8 bp Scar; No In-Frame Fusions...– Berkeley Standard (RFC 21) Prefix: GAATTC ATG AGATCT Prefix Enzymes: EcoR1 , BglII Suffix: T GGATCC ...Silver Standard (RFC 23) Prefix: GAATTC GCGGCCGC T TCTAGA G Prefix Enzymes: EcoR1 , NotI , XbaI Suffix: T...CTGCAG Suffix Enzymes: SpeI , NotI , PstI Scar: ACTAGA Features: Modified RFC 10 with shortened prefix...Freiberg Standard (RFC 25) Prefix: GAATTC GCGGCCGC T TCTAGA TG GCCGGC Alternate Prefix: GAATTC GCGGCCGC T TCTAG...

...which is immobilized on agarose or magnetic beads. Using centrifugation (for agarose beads) or a magnet (...developed, the antibodies are collected, tested against the antigen, packaged, and sold. This is the most...unknown Monomer with a valency of 2 IgE Protects against parasites and is responsible for driving allergic... directly to the sdAb, or by using a secondary against a tag, such as His, incorporated in the sdAb. sdAbs...materials. There are two primary methods used, using agarose or magnetic beads, but both rely on the same general...most or all of their extracellular matrix removed. Again, the signaling molecule may be a fluorophore or ...or more conjugated primary antibodies, and wash again. Use controls to identify, or gate, cells by size...

...the “deletion band” may still amplify), resolve agarose gel to determine if size is consistent with the....2.1 - 3.2.2 ). Plate onto a lysogeny broth (LB) agar plate with 100 μg/ml ampicillin and incubate O/N...testing the bulk sorted cells. Run samples on 2% agarose gel at 10 V/cm using 1x Tris-acetate-EDTA (TAE)...non-deletion amplicons be easily resolved on an agarose gel for multiplexing. Screening CRISPR/Cas9 Clones...plasmid into DH5α E. coli cells and plate onto LB agar plates with the relevant antibiotic. Select multiple...protein, perform an immunoblot using an antibody against the relevant protein 9 . References This protocol...CRISPR-Cas9 system. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Nature Protocols . 2013....

...RAB7 GFP Richard Pagano 12661 DsRed-rab7 WT Late endosomes RAB7 DsRed Richard Pagano 158006 pCMV-mGold-Lysosome-N... GFP Richard Pagano 12679 DsRed-rab11 WT Recycling endosomes Rab11a DsRed Richard Pagano *Fusions to other...DsRed-Rab5 WT Early endosomes RAB5A DsRed2 Richard Pagano 42307 GFP-EEA1 wt Early endosomes EEA1 GFP Silvia...

...itself, will protect the HR template from the Cas9. Again, if it's within coding region make sure it's a silent...Forward: CCATCCCCTTCTGTGAATGT EMX1-Reverse: GGAGATTGGAGACACGGAGA Why does the PX260 plasmid use 30nt oligos...

...utilized CRISPR as a powerful defensive strategy against viral invaders, and this system is proving to be...genome in 1987, but their function as a safeguard against bacteriophages was not elucidated until 2007. Scientists...Horvath P. 2007. CRISPR provides acquired resistance against viruses in prokaryotes. Science . 315(5819):1709...Cell . 154(2):442-51. PMID: 23849981 Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. 1987. Nucleotide...

...antibodies A panel of recombinant monoclonal antibodies against zebrafish neural receptors and secreted proteins...Toolbox for making synthetic nanobodies (sybodies) against membrane protein targets. Synthetic single domain...May 24;7. and Generation of synthetic nanobodies against delicate proteins. Nat Protoc. 2020 Apr 8. Markus...

...Acap3 116983 834 GAP Agap1 116987 857 GAP Agap2 116986 1192 Bacterial (pET) GAP Agap3 116988 911 Bacterial...