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Enter a sequence to perform a BLAST-based search of Addgene plasmid sequence databases.

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We narrowed to 938 results for: mpl

Showing: 901 - 920 of 938 results

  1. Protocol - How to Design Primers

    Type
    Protocol
    ...correspond to sequences on the template molecule (must be complementary to template strand). However, primers...One needs to design primers that are complementary to the template region of DNA. They are synthesized ...primers do not need to correspond to the template strand completely; it is essential, however, that the 3’...3’ end of the primer corresponds completely to the template DNA strand so elongation can proceed. Usually...well. Short primers are mainly used for amplifying a small, simple fragment of DNA. On the other hand, a... a long primer is used to amplify a eukaryotic genomic DNA sample. However, a primer should not be too...primers produce inaccurate, nonspecific DNA amplification product, and long primers result in a slower...

  2. Western Blot

    Type
    Protocol
    ...duplicate samples on the plate. Subtract the average absorbance of the blank from all of the samples. Plot...concentration of the sample from the standard curve. Determine the volume of sample required to load an...protein for each sample. Pro-Tip The ideal total protein loaded will vary between samples and target proteins...abundance in the sample you will need to load a greater amount of total protein. Prepare sample for loading...Dilute 4X protein loading buffer in the sample to 1X . Boil the samples for 10 min at 100 °C . Section 3: SDS-PAGE...blotting to visualize a protein from cells or tissue samples. Equipment Microcentrifuge 0.5–10 µL single channel... the host species of the primary antibody. For example, use an anti-mouse secondary antibody for primary...

  3. Plasmid Cloning by PCR (with Protocols)

    Type
    Protocol
    ...to the stop codon (TGA, in this example). Assuming you are amplifying from plasmid DNA (rather than from..., but we need to use the reverse complement to get PCR amplification. We can start similarly, taking ...generate the reverse-complement of this sequence so that we can successfully amplify the ORF. You can generate... to be amplified (the ORF in this case), not the Tm of the entire primer. If you are amplifying from a... a plasmid or simple template, there is very little chance for mis-priming, so you can use a pretty wide...between samples. In addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each...choice with minimal limitations. Background In its simplest form, PCR based cloning is about making a copy...

  4. Protocol - How to Run an Agarose Gel

    Type
    Protocol
    ...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 and mix well...Note: When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or...bands needed to be separated - see FAQs below . Simply adjust the mass of agarose in a given volume to...). Microwave for 1-3 min until the agarose is completely dissolved (but do not overboil the solution, ...room temperature for 20-30 mins, until it has completely solidified. Pro-Tip If you are in a hurry, the... well. Use 5 µl of loading buffer per 25 µl of sample. Note: Loading buffer serves two purposes: 1) it...

  5. Ligation Independent Cloning

    Type
    Protocol
    ...sterile dH20 to 40 μl Step 4: Amplify Insert by PCR Perform PCR amplification of your insert following the...through the transformation/replication process. LIC employs long overhangs to form a stable association between...replication cycle. Empty vectors for LIC typically employ type II restriction enzymes (e.g. BsaI), which ...counter-selection for negative clones as in the example shown here. The manufacturer of an LIC-specific...respective primers. Below we use pNIC28-Bsa4 as an example of LIC experimental design. Search Addgene's collection... Your Primers Primer design for LIC is often as simple as using the backbone manufacturer's suggested ...sequence is used, plus a minimum of 18 bp of your template sequence. 5' and 3' primers will have different...

  6. AAV Production in HEK293 Cells

    Type
    Protocol
    ... 50% amplitude. Return to ice between each round of sonication to avoid overheating of the sample. Mix... mM NaCl, 2 mM MgCl2) Reagent Preparation DMEM Complete : 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...min. Neutralize trypsin by adding 10 mL of DMEM Complete. Pipet back and forth vigorously multiple times...-175 flasks. Adjust volume to 300 mL with DMEM complete media and mix. Seed all cells in 1 CS2. Return...CS2 to help detach the cells, add 200 mL of DMEM complete media, then transfer the cells into a sterile ...sterile bottle. Rinse the CS2 with 100 mL of DMEM complete medium and pool with the cells harvested in the previous...0.08 μg/bp Therefore, for each plasmid we need: Sample Calculations RepCap: 0.08 μg/bp × 7,265 bp = 582.1...

  7. Virus Protocol - Generating Stable Cell Lines

    Type
    Protocol
    ...the lentivirus in DMEM complete + 10 µg/mL polybrene. Note, this is just a sample of possible dilutions...puromycin, blasticidin) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...kills all of the cells. Prepare a batch of DMEM complete + 10 µg/mL polybrene by diluting 20 µL of 10 mg...Dilution Volume of Lentivirus (μL) Volume of DMEM complete + 10 µg/mL polybrene (µL) 0 0 500 1:5 300 200 ...,000 cells into a total volume of 7 mL of DMEM complete + 10 µg/mL polybrene. Mix well by pipetting or...all the media in these wells was made with DMEM complete + 10 µg/mL polybrene, the final concentration ...aspirate the media from the cells. Add 1.5 mL of DMEM complete containing the appropriate antibiotic. This is...

  8. Pouring LB Agar Plates

    Type
    Protocol
    ...growth. See our sample data section below for positive and negative test results. Sample Data In all cases...bacteria containing the plasmids of interest (for example, fewer satellite colonies will grow). However, ...on the number of plates you’d like to pour. For example: Because we’d like to make 20 plates, and our plates...will darken during the autoclave process if your sample has spent at least 10 min at 121 ℃. Use lab tape...the autoclave and run on a setting that gets the sample to at least 121 ℃ under 20 psi for at least 30 ... any weird and wonderful organisms. While your samples are sterilizing in the autoclave, you should prepare... you should create a 1000x stock solution. For example: If you’ll be preparing plates with a final concentration...

  9. DNA Quantification

    Type
    Protocol
    ...Calculate your DNA sample concentration and purity using Addgene’s DNA Quantification Protocol....generally used as an indicator of the purity of DNA samples. These days, many labs have a NanoDrop, which is...lab. Spectrophotometer Tips Before measuring any samples, be sure to ‘blank’ the spectrophotometer using...your solvent. If using a NanoDrop to measure your samples, place 1-2µL of mini-prepped DNA onto the pedestal... purity ranges from 1.80-2.00). Repeat for each sample. Notes: Keep in mind that despite the accuracy ...accuracy of the NanoDrop, if two consecutive samples have significantly different concentrations, it is possible... idea to re-zero any spectrophotometer between samples if they are expected to vary significantly in concentration...

  10. Colony Formation Titering Assay

    Type
    Protocol
    ...Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...colony formation assay. Prepare a batch of DMEM complete containing 10 μg/mL polybrene by diluting 20 μL...fold serial dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene: Dilution Volume...Lentivirus or Previous Dilution (μL) Volume of DMEM Complete Containing 10 μg/mL Polybrene (μL) Volume of Virus...follows: Dilute 7,000 cells into 9.45 mL of DMEM complete containing 10 μg/mL polybrene. Mix well by pipetting...to 8 h and the media can be replaced with DMEM complete. However, antibiotic-containing media should not...aspirate the media from the cells. Add 1.5 mL of DMEM complete containing the appropriate antibiotic. Incubate...

  11. Protocol - How to Purify DNA from an Agarose Gel

    Type
    Protocol
    ...contaminating your sample. To accomplish this, it is best to skip lanes between samples and between the ...bands out of the agarose gel and purify the DNA samples. This is a commonly used technique for molecular...the ladder and nearest sample. To minimize the risk of DNA damage, it is best to limit the UV exposure of...do you get better resolution of bands? A couple simple ways to increase the resolution (crispness) of ...will help separate larger bands. 10% Rule For each sample you want to load on a gel, make 10% more volume...several microliters can be lost in pipetting. For example, if you want to load 1.0 μg in 10μL, make 1.1μg...

  12. Fluorescence Titering Assay

    Type
    Protocol
    ...biosafety level 2+. Please ensure that you are in compliance with your institution’s biosafety regulations...Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...follows: Dilute 525,000 cells into 14 mL of DMEM complete. Mix well by pipetting or inverting. Aliquot 2... Prepare dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene. Note, this protocol...Volume of Lentivirus Stock (μL) Volume of DMEM complete (μL) Volume of 10mg/mL polybrene (μL) 1:10 150...Dilution Factor V T = Transduction Volume, mL For example: If 150,000 cells were transduced in the 1:100 ...cells with fluorescence V V = Virus Volume, mL For example: If 15 µL of virus added to 150,000 cells resulted...

  13. Lentivirus Production

    Type
    Protocol
    ... 15 mL DMEM Complete. Wednesday: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM Complete. Friday: Plate...your insert of interest Reagent Preparation DMEM Complete (also known as D10): 10% v/v FBS and 4 mM L-alanyl-L-glutamine...Plate 8×10 5 cells in a T75 flask in 15 mL DMEM Complete. Do not add pen-strep to the media. Use cells ...packaging cells at 3.8×10 6 cells per plate in DMEM Complete in 10 cm tissue culture plates. Incubate the cells...at RT. During the incubation, add 10 mL of DMEM Complete to a 15 mL conical and an appropriate amount of...the media. Replace the media with 10 mL of DMEM Complete or OptiPro SFM. Incubate the cells. Virus can ... °C as soon as possible to avoid loss of titer. Sample Data Figure 1: 293T cells were transfected with...

  14. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ... kind either expressed or implied, including but not limited to the implied warranties of fitness for ...sequence), while the EcoRI site is preserved. For a complete map of pLKO.1 containing the 1.9kb stuffer, visit... nuclear import of the vector’s preintegration complex in the transduced cells. hPGK Human phosphoglycerate... set of top-scoring targets for your gene. For example, the Whitehead Institute for Biomedical Research...AATTCAAAAA—21bp sense—CTCGAG—21bp antisense 3’ For example, if the target sequence is (AA)TGCCTACGTTAAGCTATAC...boiling water. If using a PCR machine, incubate the sample at 70°C for 10 minutes then slowly cool to room...will require 6 μL FuGENE® + 74 μL OPTI-MEM. For example: 1x master mix: 6 μL FuGENE® + 74 μL OPTI-MEM 5x...

  15. Protocol - How to Perform a Diagnostic Digest

    Type
    Protocol
    ...have become the most widely used method scientists employ to selectively move a specific piece of DNA from...Plasmid Size -OR- Insert and Backbone Size The simplest form of diagnostic digest is one in which you ... verification, such as DNA sequencing . In the example above, digestion with enzyme RE1 will linearize...result in similar sized bands, thus making this simple digest less informative. This is particularly true...patterns so that you get double confirmation. In the example above, digestion with either RE3 or RE4 will give...the insert is in the correct orientation. In the example below we want to know how to differentiate between...products from each orientation are different sizes. Simply run the digest products on a gel and those clones...

  16. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ... fresh complete medium. This 5 cells/mL solution will be used to seed the 96-well plate. Sample Calculation...Corning 430167 (optional) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...performed at Addgene on the A549 cells described in our sample data , each 96-well plate gave rise to 15–20 monoclonal...Each 10 cm dish should be seeded in 10 mL DMEM complete, which will generate enough conditioned medium...adding 10 µL homogenized cell solution to 990 µL complete medium. Instead of transferring 0.125 µL, now ...transgene expression and/or other phenotypes. For example, perform Western blotting to screen for lines with...highest or lowest transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal cell lines...

  17. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...compatibly digested vector backbone. This is accomplished by covalently connecting the sugar backbone ...is physically attached to the backbone and the complete plasmid can be transformed into bacterial cells...overhanging base pairs on the vector and insert are complementary, the two pieces of DNA connect and ultimately...ultimately are fused by the ligation reaction. The example below depicts the ligation of two sticky ends that... will allow you to verify that the vector was completely digested and if phosphatase treated, that the... 3:1 ratio is not working or when doing more complicated cloning. While 3:1 will get you in the ballpark...the molarity of DNA ends available for ligation. Simply put, there are only two ends on any given piece...

  18. General Transfection

    Type
    Protocol
    ...DMEM complete containing 25 µM chloroquine diphosphate and incubate ~5 h For 10 mL of DMEM complete, add... of DMEM complete. Incubate the cells 24–48 h before checking for protein expression. Sample Data Legend...Syringes for filtering Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...HEK293T cells at 3.8x10 6 cells per plate in DMEM complete in 10 cm tissue culture plates. Incubate the cells...

  19. Transfection for Recombinant Antibodies

    Type
    Protocol
    ...be resuspended before sampling. Gently swirl the flask 5–10 times before sampling. Transfer 10 µL of trypan...biosafety level 2. Please ensure that you are in compliance with your institution’s biosafety regulations...the PEI-MAX and recombinant antibody plasmid DNA sample to the biosafety cabinet and incubate for 1 h at...significantly and should be empirically determined for your sample. Typical ratios may range from 1:1 to 1:6. Add ...supplement the flask with 3.75 mM valproic acid. Example feeding schedule: Thursday: Transfect cells. Friday...

  20. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    ...Add your sample. Spin at 3500 rpm for 5–8 min at 4 °C, discard the flow through. Add more sample and spin...exchange and concentration Note: Both steps could be completed in one (long) day. Equipment Ultracentrifuge T70i...After each spin, add more formulation buffer and sample and make sure to pipet back and forth a few times..., or aliquot and store at -80 °C for long term. Sample Data Figure 1: Iodixanol gradient before ultracentrifugation...opens in a new window) copyright (2006). Figure 3: Example of an SDS-PAGE gel of 10 consecutive gradient fractions...
Showing: 901 - 920 of 938 results