...systems utilize with type I-F Cascades. Figure 2: Cascade complex from CRISPR type I-E. Created with...using Cas9, two or more gRNAs must be used along the same chromosome. During DNA repair, there is a chance...

...of concept for further development. Figure 2: Mechanisms of deaminase-based adenine transversion...instead of human AAG (remember, MPG and AAG are the same protein!) (Chen et al., 2024). They called their...different teams succeeded with this approach around the same time (He et al., 2024; Tong et al., 2024; Ye et ...hypoxanthine from DNA, creating an abasic site. The same protein (or its homologs) has more than one name...

...would help them grasp some of the key concepts (Fig. 2). One thing I will likely change for next time is ...essentially background slides getting everyone on the same page in terms of the vocabulary--DNA, protein, plasmid...

...that view (Callahan et al., 2022).     Fig. 2: Students in an MDH CURE replaced select residues ...happened in the lab, and he wanted to provide the same kinds of opportunities for his students. But he ...

... Methods 2009; 6, 343-345. PubMed PMID: 19363495. 2. Wang JW, Wang A, Li K, Wang B, Jin S, Reiser M, Lockey... mix of enzymes is that they can all work at the same temperature, so the entire reaction takes an hour... to complete at 50 °C. After an hour or so, the sample is immediately ready to transform into competent... generate wild type and mutant constructs at the same time, rather than sequentially.   How does Gibson...

...the necessary steps to perform:      Fig. 2: Quick-stepping to a FASTA file   With our outline...knew we could rely on the containers to produce the same output regardless of who or which computer ran the...

...mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR cassette is flanked by two direct... introduce multiple genetic modifications in the same strain. MazF expression is under control of the ...

...PMID: 18478103. PubMed Central PMCID: PMC2364656. 2. Dittmar, Kimberly A., et al. "Selective charging ...producing additional tRNAs is that you can use the same expression system for many different genes without...

...) or the American Chemical Society (ACS) are just 2 examples of organizations that hold large, annual ...stand out from the hundreds of others that did the same thing? Make an effort to meet people. Attend smaller...

...Single-chain variable fragment (ScFv) Figure 2: Comparison between the IgG antibody and scFv. ... exact amount of protein in your sample, you’ll compare your samples to a standard curve. This curve is...the known samples is used to calculate the concentration of protein in the experimental samples. Get more...multiple proteins on the same cell simultaneously as well as compare between samples. Learn more about flow...of different isotypes can be used together in the same experiment because the reagents used to detect the... population of antibodies that all recognize the same epitope of the target protein. Most commonly these...expression in cell lysates, whole cells, or tissue samples. Many experiments that use antibodies use a primary...

...to the break room for a sip of water.  Fig 2: Blugene likes to spark creativity in out on the open...you and you will arrive at unique solutions to the same problem. If your lab/company/team is still working...

...positive cells (Martin et al., 2019). Figure 2: Editing efficiencies using base editing reporters...by canonical base excision repair enzymes. At the same time Cas9n cleaves the opposing DNA strain which...

...gene1 [lon] ompT gal sulA11 R(mcr-73::miniTn10--TetS)2 [dcm] R(zgb-210::Tn10--TetS) General protein expression...-deficient; allows for cloning and expression in same strain F- recA1 hsdR(rK12- mK12+) (DE3) (RifR) ...

... PMID: 9537413. PubMed Central PMCID: PMC3010411. 2. Kohli, Manu, et al. "Facile methods for generating...require modifying multiple genes simultaneously in the same cell, are more appropriate for a CRISPR-based approach...

... Watch Joanne give her "Not" Networking 101 Talk 2. Tailor every talk to the occasion Don’t just plan... it perfectly fits the occasion.  I speak on the same topics many times, but I always seek out information...

...Postdoctoral Associations around Boston - From Panel Session 2: Defining the Postdoc: An Institutional Perspective...underrepresented groups under their mentorship with the same critical eye they place on others within their labs...

...improvement in muscle physiological performance.  2. Trans-splicing Overview: For trans-plicing, splice...nbt0702-697 Hirsch ML, Agbandje-McKenna M, Jude Samulski R (2010) Little Vector, Big Gene Transduction:...Bellon I, Yin C, Chavala S, Pryadkina M, Richard I, Samulski RJ (2013) Oversized AAV Transductifon Is Mediated...

...variety of environmental conditions, and double every 2 hours. Yeast are also the first eukaryotic genome ... model organism in scientific research. Yes, the same kind we use in breads and other baked goods! Yeast...

..., whereas the mVenus-APT1 fusion does not (figure 2). In the mVenus-APT1, a lipidation motif, which is...conclusive evidence that the fusion operates in the same way as the native protein. Although a fluorescent...

... iRFP713, % Oligomeric state Photo-stabilityc, t1/2, s pKa Brightness in HeLa cells vs. iRFP713, % d ..., and deep penetration of red-shifted light. The same NIR FPs and biosensors can be imaged at spatial ...

...Nutr, 129 (2009a): 477–484. PubMed PMID: 10064313. 2. Chapman-Smith, A., & Cronan, J. E. “The enzymatic...pre-incubated with free avidin can be added to your sample. Any free biotin-binding sites on this complex ...

... more from the authors in this blog post. Figure 2: iGluSnFR3 displays enhanced brightness, expression...to the range of concentrations expected in your samples. Higher affinity isn’t always better, especially...

...15.4%) 46 (59%) of these students were in the last 2 years of their degree. Students filled in a pre-program... my mentor, since she does research at UK in the same field that I do: prostate cancer. She has guided...

...biology 14.6 (2004): 757-764. PubMed PMID: 15582400. 2. Tomkinson, Alan E., et al. "DNA ligases: structure...Watson-Crick base pair, are joined with nearly the same efficiency as a correct base pair. DNA ligases have... resistance-mediating polymorphisms in African samples of Plasmodium falciparum." Journal of clinical ...

...experience in cancer research After graduating with a 2:1 BSc in Molecular Biology (roughly a B average in...cancer and I loved the practical aspects of testing samples in the lab, but I really struggled with the large...

...effective recovery of knock-ins, but did result in 1-2 kilobases of additional sequence being introduced....changes 5’ to the PAM, sequences corresponding to the same strand as the PAM produced a higher editing efficiency...

...indicated, the antibiotic powder can be dissolved in dH 2 0. Addgene recommends making 1000X stock solutions...daughter cells. These daughter cells contain the same genetic information as the parental cell, and are...

... 10 90 2 x 10 8 10 of 2 x 10 8 dilution 90 2 x 10 7 10 of 2 x 10 7 dilution 90 2 x 10 6 10 of 2 x 10 6...6 dilution 90 2 x 10 5 10 of 2 x 10 5 dilution 90 2 x 10 4 10 of 2 x 10 4 dilution 90 2 x 10 3 Pro-Tip...molecules/μL To obtain a solution at 2 x 10 9 molecules/μL: 1.59 x 10 11 / 2 x 10 9 = 79.8X dilution ...your standard curve plasmid (2 x 10 9 stock made in step #1): Volume of 2 x 10 9 stock or previous dilution...stock 45 uL 10X 10X Dilution 2 5 uL Dil. 1 95 uL 20X 200X Dilution 3 20 uL Dil. 2 80 uL 5X 1000X Dilution ...empty NTC C AAV reference Sample 3 D E Sample 1 Sample 4 F E Sample 2 Sample 5 F Perform data analysis...virion). 5 μL sample + 39 μL H 2 O + 5 μL 10X DNase buffer + 1 μL DNase Gently mix sample (do not vortex...

...phosphate monobasic monohydrate (NaH 2 PO 4 ∙H 2 O), pH 7.0 138 g NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust...sodium phosphate dibasic (NaH 2 PO 4 ), pH 7.0 142 g of NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust pH...monobasic monohydrate (NaH 2 PO 4 ∙H 2 O) 610 mL of sterile sodium phosphate dibasic (NaH 2 PO 4 ) Store up to...purifying the same recombinant antibody. Section 2: Buffer exchange Choose Option 1 or Option 2 based on the...antibody sample. Pro-Tip If the volume of the sample is greater than 4 mL , then divide it into 2 columns...concentration of the pooled sample is below 1.0 mg/mL proceed to Option 2 with a buffer exchange/concentration...Centrifuge at 1000 x g for 2 min to collect the sample. Discard column after use. Determine antibody concentration...

...430825, 175 cm 2 Cellstack 5, Corning 3319, 3180 cm 2 Cellstack 2, Corning 3269, 1272 cm 2 Heat-inactivated...: 50 mM Tris HCl, 150 mM NaCl, 2 mM MgCl 2 Add the following to the 2 L sterile bottle: 1836 mL deionized... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working... mL of PBS. Aspirate PBS and add 2 mL of 0.05% Trypsin/EDTA. Wait ~2 min. Neutralize trypsin by adding...(CS5) (Link opens in a new window) (3,180 cm 2 - the same surface area as 21 x T-175 flasks). Cell stacks...flasks. Workflow Timeline Day 0: Seed cells in CS2 Day 2: Seed cells in CS5 Day 3 (am): Transfect cells Day...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...

...Activation 95 10 2 1 Denaturation 94 0.5 2 40 Annealing/Extension 60 1 2 40 Enzyme Deactivation 98 10 2 1 Hold ...diploid cells, thus the reason for multiplying by 2. $$V = 2*{copies\ RRE \over copies\ RPP30}$$ Use the viruses...integrated copies of RRE. Since the samples that are assayed are diluted 2-fold serially, the concentration...factor of 2 across the dilutions. RPP30 copies should be relatively constant across samples. In the RRE...RRE example below, 2-fold serial dilutions of a sample were loaded in wells B01-H01. As shown in the image...Figure 2) below, the concentration of RPP30 positive droplets stays relatively even across samples (green...ddPCR Lentivirus sample data, RRE positive (blue) and negative (black) droplets Figure 2: ddPCR Lentivirus...

...materials D.2 Screening for inserts E. Producing Lentiviral Particles E.1 Recommended materials E.2 Protocol...Published articles H.2 Web resources I. Appendix I.1 Sequence of pLKO.1 TRC-Cloning Vector I.2 Recipes I.3 Warranty...VWR: #7177-48-2. Use at 100 μg/mL. Carbenicillin VWR: #80030-956. Use at 100 μg/mL. C.2 Annealing Oligos...oligo 5 μL Reverse oligo 5 μL 10x NEB buffer 2 35 μL ddH 2 O Incubate for 4 minutes at 95°C in a PCR machine...buffer 1 1 μL AgeI add ddH 2 O to bring to 50 μL final volume Incubate at 37°C for 2 hours. Purify with Qiaquick...buffer for EcoRI 1 μL EcoRI 14 μL ddH 2 O Incubate at 37°C for 2 hours. Run digested DNA on 0.8% low melting... For a standard T4 ligation, mix: 2 μL annealed oligo from step C.2 20 ng digested pLKO.1 TRC-cloning ...

... Example for AR0018 (lane 2 in Figure 1): Sample Peak 1 (contaminant) Peak 2 (contaminant) Peak 3 (HC)...follows: Figure 2 Using the box tool, draw a box around the entire first gel lane (as in Figure 2). Select Analyze...choose Use Equation . Select the Show R 2 checkbox. Pro-Tip The R 2 of the trendline should be between 0.95...of 4X sample buffer to each sample. Add 2 µL 10X reducing agent to each sample. Spin the sample briefly...Equipment Heat block 1–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...bottom part of the gel where dye is visible. Section 2: Staining the Gel Place the gel in a plastic tray .... Figure 1 Recombinant antibody preps should have 2 clear bands at ~50 kDa and ~25 kDa corresponding to...

... 95 10 2 1 Denaturation 95 0.5 2 50 Annealing/Extension 60 1 2 50 Signal Stabilization 98 10 2 1 Hold ...factor of 2 across the dilutions. In the example below, 2-fold serial dilutions of a sample were loaded...AAV sample, and the three final dilutions are assayed. The samples that are assayed are diluted 2-fold... considered biosafety level 1 but may require BSL-2 handling depending on the insert. Please ensure that...single channel pipette 1–10 µL multichannel pipette 2–50 µL multichannel pipette 20–200 µL multichannel ...20X): 5 µL in 95 µL 1X PCR buffer (1:20) Dilution 2 (20X): 5 µL in 95 µL 1X PCR buffer (1:400) Dilution... DG8 cartridge into the cartridge holder. Using a 2–50 µL multichannel pipet, load 20 µL of the reaction...

...PCR tube Ice Bucket 2 μL Template DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 ... reagents on ice): 2 μL Template DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 ...normally sterile dH 2 O. To make a 100uM stock of any primer, add a number of µl of dH 2 O equal to the number...annealing temperature step-wise by 1-2°C. The rate of DNA synthesis is ~1-2 kb/min. The extension time can ...work adequately. Divalent cations such as Mg 2+ and Mn 2+ stabilize the buffer solution. These cations...(PCR). Basic PCR Program Initial Denaturation for 2 minutes at 94°C: This initiation step heats the double...target DNA strand accurately and rapidly. Repeat steps 2-4 25-30 times. Final Extension for 5 minutes at 72...

...cells or tissue samples. Equipment Microcentrifuge 0.5–10 µL single channel pipette 2–20 µL single channel...transfer, block, incubate with primary antibody Day 2: Incubate with secondary antibody Video Watch this...Heat block Mini gel tank chamber Power supply iBlot 2 Gel Transfer Device Roller Spatula Platform shaker...running buffer Prestained protein ladder Ethanol iBlot 2 PVDF Mini Stack, Thermo Fisher IB24002 20X TBS Tween...immediately or store at -80 °C until ready to use. Section 2: Determine the total protein concentration and prepare...in deionized water. To prepare 20% ethanol, dilute 2 mL of ethanol into 8 mL of deionized water and mix...the transfer sandwich as follows: Unseal the iBlot 2 PVDF Mini transfer stack. Set the Top Stack to one...

... °C for short term (2 weeks), or aliquot and store at -80 °C for long term. Sample Data Figure 1: Iodixanol...purification. Workflow Timeline Day 1: Purify Day 2: Buffer exchange and concentration Note: Both steps... 7.4 1X PBS-MK buffer 100X Pluronic-F68 NaCl MgCl 2 KCl Centrifugal filter units (MWCO 100 kDa) Reagent...PBS-MK buffer Dissolve 5.84 g of NaCl, 26.3 mg of MgCl 2 and 14.91 mg of KCl in 1× PBS in a final volume of...at 4 °C. 1X PBS-MK buffer Dissolve 26.3 mg of MgCl 2 , and 14.91 mg of KCl in 1× PBS in a final volume ...(C) (formulation buffer) Add 5 mL of Buffer B and 2 mL of 5 M NaCl to 43 mL PBS Procedure Preparation ...need more time, you can alternatively centrifuge for 2 h at 200,000 x g at 18 °C. Carefully take the QuickSeal...

...created in section 1, step 2 is a suggested starting point. If your unknown sample’s absorbance falls above...vapors. Workflow Timeline Day 1: Antigen Coating Day 2: Blocking Day 3: Primary antibody incubation Day 4...with 96-well plates 1–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...serial dilutions of the purified antigen as follows: 2 ng/µL : Add 100 µL of 20 ng/µL stock into 900 µL PBS...microfuge tube and vortex. 1 ng/µL : Add 450 µL of 2 ng/µL stock into 450 uL PBS in a microfuge tube and...37 °C for 30 min , or overnight at 4 °C . Section 2: Block the plate Prepare the wash buffer (0.05% Tween...incubate on a microplate shaker set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section...

... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working...protein expression. Sample Data Legend: Lenti-X 293T cells were transfected using 1:1, 1:2, 1:3 and 1:6 µg... for viral production) Day 1: Transfect Cells Day 2 (am): 18 h post transfection - Remove media, replace...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container... use. Thawed aliquots should be discarded after 1–2 months. 1 mg/mL polyethylenimine, linear MW 25,000...times a week: Monday: Plate 1x10 6 cells in a 75 cm 2 flask in a volume of 15 mL. Wednesday: Plate 1x10 ...

... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working...μg total DNA to μg PEI ratios of 1:1, 1:2, 1:3 and 1:6. The 1:2 and 1:3 total DNA:PEI μg ratios provided...packaging cells Day 1 (pm): Transfect packaging cells Day 2 (am): 18 h post-transfection. Remove media, replace...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...culture plates. Incubate the cells at 37 °C, 5% CO 2 for ~20 h. Prepare a mixture of the three transfection...plasmid using a variety of ratios. Check the cells 1-2 days after transfection to determine what ratio gives...

...Warnings HEK293 cells are considered biosafety level 2. Please ensure that you are in compliance with your... 18, 2022 Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3-6: Feed cells Day 7: Harvest...conical tubes Automated cell counter 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm 37 ...250 mg Benzamidine 25 mL Aprotinin saline solution (2 mg/mL) Mix well and sterilize through a 0.2 µm PES... a 500 mL vented flask.Incubate in a 37 °C, 5% CO 2 incubator on a shaking platform set to 120 rpm. Pro-Tip... not use cells that are over 30 passages. Section 2: Transfection Check the cell density and viability...the culture. Pro-Tip Culture should be between 1.5–2 x 10 6 cells/mL with >95% viability to proceed with...

...RNAzol®, QIAzol® (for Protocol Option #2) Water-saturated Phenol 2 M Sodium Acetate pH 4 Chloroform/Isoamyl...entire RNA sample, consider making smaller aliquots of it and storing those in -80°C. Option #2 - TRIzol...vigorously by hand for 10 seconds. Incubate the sample(s) for 2-3 minutes on ice and centrifuge for 15 minutes....5% (wt/vol) N-laurosylsarcosine (Sarkosyl) 0.1 M 2-mercaptoethanol TRIzol® or similar product such as...recipe). If using TRIzol®, jump down to the Option #2 - TRIzol® Protocol section below. Homogenize or lyse...following sequentially to 1 mL of lysate: Add 0.1 mL of 2 M sodium acetate (pH 4.0), mix thoroughly by inversion... 10 seconds. Incubate sample(s) for 15 minutes on ice and centrifuge the sample(s) for 15 minutes at 12,000...

... glacial acetic acid 57 mL of dH 2 O Store Solution III at 4°C Grow 2 mL overnight cultures from single...Resuspension buffer Denaturing solution Renaturing solution 2 mg/mL RNase A TE or water-saturated phenol-chloroform...100 μL of cold Solution I. Vortex the solution for 2 min or until all bacteria are fully resuspended. Add...pipetting or carefully pouring. Optional: Add 5 μL of 2 mg/mL RNase A to the supernatant in the new tube and... to the recovered aqueous DNA layer. Repeat steps 2-4. Note: Phenol-chloroform is a hazardous waste - ...: Ethanol Precipitation To your DNA solution, add 2-2.5 volumes 95% or 100% ethanol and 1/10 volume of...of DNA Samples Add an equal volume of TE-saturated phenol-chloroform to the aqueous DNA sample. Pro-Tip...

...method 1) or the volume of virus (method 2): Method 1 Method 2 $$T = {N*F*D\over V_T}$$ Where: T = Titer...Lentivirus is generally considered biosafety level 2+. Please ensure that you are in compliance with your...Day 0: Seed 293T cells Day 1: Transduce cells Day 2 (am): Remove media, replace with fresh media Day 4...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...complete. Mix well by pipetting or inverting. Aliquot 2 mL of cell suspension into each well of the 6-well... titer, take the average of multiple dilutions. Sample Data Figure 1: 293T cells were transduced with ...

...agarose concentrations (e.g., 2 g of agarose in 100 mL of TAE will make a 2% gel). Mix agarose powder with...migrated; 2) it contains a high percentage of glycerol that increases the density of your DNA sample causing...gels are commonly used in concentrations of 0.7% to 2% depending on the size of bands needed to be separated...concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds...steadily, push the sample out and watch as the sample fills the well. After all of the sample is unloaded, .... Loading Samples and Running an Agarose Gel: Add loading buffer to each of your DNA samples. Note: Loading...Note: When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or...

...Workflow Timeline Day 0: Seed and transduce cells Day 2: Replace media with fresh media containing selection...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...the appropriate antibiotic. Incubate the cells for ~2 weeks. All of the cells in the untransduced (negative...disturb the colonies. Count the colonies for at least 2 of the dilutions. Pro-Tip The higher dilution wells...stained with 0.1% crystal violet and counted. Figure 2: A549 cells were transduced with the indicated serial...protocol outlines the seeding of the cells at the same time as the virus-mediated transduction. For some...

...highest or lowest transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal cell ...Day 1: Seed individual cells in a 96-well plate Day 2–14: Monitor cells for growth and expand cells Day ...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...channel pipette 200–1000 µL single channel pipette CO 2 incubator Pipet controller Hazardous waste container...approximately 50–60% confluent. For 293T cells this is about 2 × 10 6 cells in a 10 cm dish. Each 10 cm dish should...inferred by the differences in colony size. Figure 2: Cas9 expression in monoclonal cell lines generated...performed at Addgene on the A549 cells described in our sample data , each 96-well plate gave rise to 15–20 monoclonal...

...Fluorescent microscope 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... plate 4% Paraformaldehyde 5 mg/mL 4′,6-diamidino-2-phenylindole (DAPI) Bovine serum albumin (BSA) Triton...grow to the desired density before labeling. Section 2: Fixing and permeabilizing cells Gently aspirate the...of the diluted antibody to the wells and incubate 2 h at RT . Remove the primary antibody and dispose ...optimal fixation method will vary depending on the sample type and the protein of interest. You may need ...expected and specific, include a positive control sample that you know expresses the protein, such as cells...the protein of interest, and a negative control sample such as cells that do not express the protein of...

...Workflow Timeline Day 0: Seed and transduce Cells Day 2–3 (am): Remove media, replace with fresh media containing...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...cell death, the cell media should be changed every 2–3 days to maintain the dose of antibiotic, which may...confluent 10 cm dish can be expanded into two 75 cm 2 flasks, etc. Pro-Tip This selection method results...complete + 10 µg/mL polybrene. Note, this is just a sample of possible dilutions. You may want to try higher...monoclonal lines from the early polyclonal populations. Sample Data Figure 1: Generation of monoclonal cell lines...

... recover, set up overnight growth (Estimated time 2-3 hours) Transformation should be performed at the... day to ensure that growth times are limited. Day 2: Harvest cells and purify DNA (Estimated time 3-4 ... Tubes should contain a total of 5 mL (3 mL SOC + 2 mL transformed Endura from two separate transformations...has been absorbed by the agar. This usually takes 1-2 minutes. Critical Be careful not to rip or shred the...incubation if needed. Place 100 mL sterile LB at 4 ℃. Day 2 (morning) Before beginning, prechill at least four...pellet. The total weight of each pellet should be ~1-2 g. Pro-Tip Make sure to weigh the empty tube beforehand...gel extraction of either the original sample or the amplified sample, followed by reamplification of the...