...recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere on the plasmid Will...sites to your recipient plasmid : You can modify the MCS of your recipient plasmid using Annealed-oligo ...material. We recommend 1.5-2μg of donor plasmid and 1μg of recipient plasmid. It is also critical that as much...re-circularization of the recipient plasmid. You should treat your digested recipient plasmid with a phosphatase prior...ligations for each plasmid you are trying to create, with varying ratios of recipient plasmid to insert. It...recipient plasmid + insert plate should have significantly more colonies than the recipient plasmid alone ... on your recipient plasmid alone plate, you can try ligating the recipient plasmid alone in the presence...

...for verifying plasmids like this is "plasmid fingerprinting", where you cut the plasmid into 3-8 pieces...used to verify your plasmid. The goal of a diagnostic digest is to cut your plasmid into specific sized...insert and the 5000bp backbone. Verifying Plasmid Identity by "Plasmid Fingerprinting" Often, it will be enough...for how to perform a diagnostic digest to verify plasmid size, verify insert orientation, and more. Protocols...Digest You may also like... Restriction Digest of Plasmid DNA Agarose Gel Electrophoresis Introduction Restriction...selectively move a specific piece of DNA from one plasmid to another. A diagnostic restriction enzyme digest...called restrictions sites. Often, the size of the plasmid insert and vector backbone are known and thus this...

... Protocols Plasmid Cloning by PCR Plasmid Cloning by PCR You may...recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere on the plasmid. Bonus...recipient plasmid. Remember to insert your DNA in the correct orientation in the recipient plasmid by viewing... 1μg of recipient plasmid. It is also critical that as much of the recipient plasmid as possible be cut...ligations for each plasmid you are trying to create, with varying ratios of recipient plasmid to insert. It...recipient plasmid + insert plate should have significantly more colonies than the recipient plasmid alone ... on your recipient plasmid alone plate, you can try ligating the recipient plasmid alone in the presence...

...Storage of Plasmids Recovering Plasmid DNA from Bacterial Culture Background Information Plasmids can carry...copy regardless of plasmid size. See the plasmid's info page to determine if your plasmid is high or low ...resistance gene on a plasmids allows researchers to easily isolate bacteria containing that plasmid from bacteria... isolate your plasmid DNA from the bacterial culture by following Isolating Your Plasmid DNA . Antibiotic... low copy plasmids? The copy number refers to the number of copies of an individual plasmid within a single...number plasmids should only need to be grown for 12-16 hr on average. Certain features of a plasmid may ...clonal) colonies of bacteria carrying a specific plasmid. However, a liquid culture is capable of supporting...

...Recovering Plasmid DNA from Bacterial Culture Recovering Plasmid DNA from Bacterial Culture You may also...concentrated plasmid DNA. This page will discuss the general procedure for purifying plasmid DNA from bacterial...perform plasmid purification without using a kit, you can find a protocol for kit-free plasmid mini-prep... the plasmid-containing supernatant. Add either ethanol or isopropanol to precipitate the plasmid DNA....high concentration of clean plasmid DNA. To improve the purity of plasmid DNA purified without a kit it...How to purify plasmid DNA from a bacterial culture, including protocols, tips, and FAQ Protocols... a new window) Promega sell kits for isolating plasmid DNA in quantities as low as a few micrograms to...

... also like... Plasmid Cloning by Restriction Enzyme Digest Restriction Digest of Plasmid DNA DNA Ligation...both storing and replicating plasmids. Because of this, nearly all plasmids (even those designed for mammalian...that will help to maintain the plasmid without rearrangement of the plasmid DNA. Additionally, specific ...tube of plasmid DNA and just need to transform bacteria so that you can grow up more of the plasmid) you ...efficient at taking up larger plasmids. If you need to transform large plasmids, it is a good idea to use ...Learn how to transform E. coli with your plasmid of interest. Protocols...introduced into a cell. Transformation of bacteria with plasmids is important not only for studies in bacteria ...

...stocks of their plasmids. This way, when you want to make more plasmid DNA, the plasmid will already be... Storage of Plasmids Creating Bacterial Glycerol Stocks for Long-term Storage of Plasmids You may also...important for long-term storage of plasmids. Although you can store your plasmid DNA at -20°C, many labs also...also like... Bacterial Transformation Recovering Plasmid DNA from Bacterial Culture Background Information... and growth temperature, can be found on your plasmid's information page. The next day you will be able...able to start an overnight culture for plasmid DNA prep the following day. Tips and FAQ The optimal concentration... glycerol stock the same time you prepare your plasmid DNA. In the morning, when you retrieve your liquid...

...flank the regions of your plasmid that you wish to confirm. Addgene's plasmid information pages recommend...Sequences" link on the Plasmid Information Pages. Addgene sequences the plasmid to verify tags, mutations...the entire plasmid. Addgene strongly recommends that you sequence any portion of the plasmid that is important...sequence analysis of a plasmid Protocols... Protocols Sequence Analysis Sequence Analysis of a Plasmid You may also like...Introduction Sequence verification of important plasmid features (such as the gene/insert, fusion proteins...common primers that are often found within the plasmid backbone (See Addgene's Sequencing Primers for ...

...complicated procedures. Plasmid Cloning Protocols for constructing and analyzing your plasmids. Virus Protocols...Amplify CRISPR pooled-plasmid libraries Diagnostic Restriction Digest Verify a plasmid using restriction ... that can be used for basic molecular biology, plasmid cloning, and titering and testing your viral preparations...Bacterial Glycerol Stocks Store bacterial strains or plasmids for long term use Watch the Video! DNA Purification...spectrophotometer Restriction Digests Cut and analyze plasmid DNA with restriction enzymes Agarose Gel Electrophoresis... the Video! Sequence Analysis Verify important plasmid features using sequence analysis RNA Extraction...from tissues or cells without a commercial kit Plasmid Cloning Name Description (Link opens in a new window...

...differences, and plasmid recombination can all impact the representation of individual plasmids in the pooled... ). Lentiviral plasmids can recombine between their LTRs resulting in a smaller plasmid containing only...protocol to perform amplification of CRISPR pooled plasmid libraries in Escherichia coli cells. Protocols... protocol allows the amplification of a pooled-plasmid library in Escherichia coli cells. Pooled libraries...libraries contain tens to millions of different plasmids in a single sample. They are often used for screening...throughput multiplexed experiments. These pooled plasmids differ by only a short region of DNA: guide RNAs...results for libraries up to 200,000 individual plasmids and can be scaled according to the actual number...

...the assay multiple plasmids containing ITR were tested. Plasmid #59462 is one plasmid that gave reliable... calculated using this plasmid, but will change if you use a different plasmid. Sample Calculation Size...Size in bp of Addgene plasmid #59462 : 6208 bp Concentration of Addgene plasmid #59462 : 1.07 μg/μL Molecular...consistent results. Use the recommended plasmid, or test multiple plasmids to find a suitable one. Some labs...ITR primer, 5'-CGGCCTCAGTGAGCGA ITR-containing plasmid for standard curve RNase-free DNase 10X DNase buffer...Nuclease Free Water 4.7 μL 470 μL Procedure Prepare a plasmid stock of 2 x 10 9 molecules/μL to generate a standard...standard curve: One option is to use plasmid #59462 from Addgene. The values highlighted below in red ...

... Protocols Restriction Digest of Plasmid DNA Restriction Digest of Plasmid DNA You ...digest of plasmid DNA Protocols...You may also like... Recovering Plasmid DNA from Bacterial Culture Purifying DNA from an Agarose Gel DNA...is also used to quickly check the identity of a plasmid by diagnostic digest . Last Upload: Oct. 11th, ...chamber Pipetman Reagents Liquid DNA aliquot of your plasmid of interest (see below for recommend amounts) Appropriate...Procedure Select restriction enzymes to digest your plasmid. Pro-Tip To determine which restriction enzymes... it isn't methylation sensitive . Plasmids grown in Dam or Dcm methylase positive strains...

...Agar Plates Bacterial Transformation Recovering Plasmid DNA from Bacterial Culture Introduction If you ...or stab culture of bacteria and need to purify plasmid DNA from it, you will want to isolate an individual...will minimize the chance of having a mixture of plasmids in your purified DNA. This protocol explains how...antibiotic. Label the bottom of the plate with the plasmid name and the date. It is also a good idea to add...overnight (12-18 hours) at 37 °C. Pro-Tip Some plasmids or bacteria need to be grown at 30 °C instead ... 37 °C. This is often true for large unstable plasmids, which sometimes recombine at 37 °C. Be sure to...single colonies, you can proceed to Recovering Plasmid DNA or use the individual colonies for other experiments...

... 0.72 pmol Transfer Plasmid* 1.64 pmol OptiPro SFM to total volume 500 μL *Plasmid concentrations and ... each transfer plasmid. Pro-Tip Endotoxins can inhibit transfection; therefore, plasmid DNA purification...endotoxin removal step. For high quality plasmid DNA, the plasmids should also be propagated in an endonuclease... cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid DNA 27.8 μg), this would be...~20 h. Prepare a mixture of the 3 transfection plasmids into OptiPro SFM: Reagent Amount per 10 cm dish...is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1-2 ...cells were transfected with the GFP-expression plasmid pRosetta using μg total DNA to μg PEI ratios of...

...Design your plasmid and order primers (see figure to the right). When designing your plasmid, think about...correct plasmid product by restriction digest . Sequence the important regions of your final plasmid, particularly...efficient and effective method for the assembly of plasmids, and molecular biologists now use this method ...segments you will need to join to create your final plasmid. Adjacent segments should have identical sequences... background and enrich for correctly assembled plasmids by designing primers to split an antibiotic resistance...reaction if you're only making a small change in a plasmid (such as point mutations). Generate DNA segments...

...How to Ligate Plasmid DNA... Protocols DNA Ligation DNA Ligation You may also like... Plasmid Cloning by Restriction...Restriction Enzyme Digest Restriction Digest of Plasmid DNA Bacterial Transformation Background Information...final step in the construction of a recombinant plasmid is connecting the insert DNA (gene or fragment ...physically attached to the backbone and the complete plasmid can be transformed into bacterial cells for propagation... and verifies the antibiotic resistance of the plasmid Cut vector - Background due to uncut vector Cut... Any colonies indicate contamination of intact plasmid in ligation or transformation reagents Optimizing...

...Storing and Handling Addgene Plasmids What to do after you receive your plasmids from Addgene Instructions...Protocol Diagnostic Restriction Digest Verify a plasmid using restriction enzymes Diagnostic Restriction...Instructions for Storing and Handling Plasmids Genomic Deletions with CRISPR Generate genomic deletions in mammalian...Transformations We walk you through a standard plasmid transformation protocol and offer some tips and...Center How to Deposit Your guide to depositing plasmids with Addgene Depositing Page Career Videos (Link...

...μg pLKO.1 shRNA plasmid 750 ng psPAX2 packaging plasmid 250 ng pMD2.G envelope plasmid to 20 μl serum-...to vary the ratio of shRNA plasmid, packaging plasmid, and envelope plasmid to obtain the ratio that gives...shRNA. psPAX2 Packaging plasmid for producing viral particles. pMD2.G Envelope plasmid for producing viral... also be used with packaging plasmid pCMV-dR8.2 dvpr and envelope plasmid pCMV-VSVG from Robert Weinberg....1 Protocol pLKO.1 - TRC Cloning Vector Addgene Plasmid 10878. Protocol Version 1.0. December 2006. Copyright... RNAi Consortium A.2 Map of pLKO.1 A.3 Related plasmids B. Designing shRNA Oligos for pLKO.1 B.1 Determine...Puromycin resistance gene for selection of pLKO.1 plasmid in mammalian cells. sin 3’LTR 3’ Self-inactivating...

...Agar Plates Bacterial Transformation Recovering Plasmid DNA from Bacterial Culture Introduction This protocol... plate and select transformed cells containing plasmids differing in their resistance genes, as one does...or other antibiotic) E. coli transformed with a plasmid containing the carbenicillin (amp) resistance gene...effective at selecting only bacteria containing the plasmids of interest (for example, fewer satellite colonies...transform DH5α E. coli cells by heatshock with the plasmid of interest. See our transformation page for a ...

...of each plasmid needed to have a 1:1:1 molar ratio with 2 mg total DNA per CS5 Plasmid Plasmid Size (bp...conditions must be optimized. Plasmids for transfection: pHelper; pRC (Rep-Cap), plasmid expressing your gene ...molar ratio of each plasmid: 2000 μg / 24,961 bp = 0.08 μg/bp Therefore, for each plasmid we need: Sample ... 1.00 727.6 pHelper 11,854 1.00 1,185 Transfer Plasmid 5,842 1.00 584.2 Total bp 24,961 In total, we would... the total number of base pairs from all three plasmids, we can determine the total μg/bp we need to achieve...Needed: 949.8 μg / 1.0 μg/μL = 949.8 μL Transfer Plasmid: 0.08 μg/bp × 5,842 bp = 468.1 μg Volume Needed... 2% HI-FBS into a sterile 1 L bottle. Add each plasmid DNA into the bottle containing the Opti-MEM. Mix...