Skip to main content
Addgene

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

Information

In Category

We narrowed to 79 results for: plasmid cloning by pcr

Showing: 1 - 10 of 79 results

  1. Plasmid Cloning by PCR

    Type
    Blog Post
    Published
    March 29, 2016, 2:30 p.m.
    ...into a recipient plasmid. Designing primers for PCR based cloning     The basic PCR primers for molecular...TGCTTAGCGGCCGCTCAGTACTTCGAGATATGCCA-3’. Preparing the PCR product for cloning Run the PCR reaction Run PCR to amplify your insert DNA... to clone from genomic DNA, a cDNA library, or by RT-PCR. Isolate the PCR product Isolate your PCR product...recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere on the plasmid Bonus... rest of the PCR reaction using a kit, such as the QIAquick PCR Purification Kit. The PCR product is now...Learn About Other Cloning Techniques Catch Up on All Things Plasmid with Our Plasmids 101 Series Get Tips...In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same time...

  2. FastCloning: A Newer, Simpler Cloning Technique

    Type
    Blog Post
    Published
    Nov. 1, 2016, 2:30 p.m.
    ... Blog Plasmid Cloning by PCR Perform Site Directed Mutagenesis by PCR Catch up on Your Plasmid Background...these include, TA cloning, ligation independent cloning, TOPO cloning, one step cloning, and overlap extension...Standard Restriction Cloning and Fast Cloning Requirement Restriction Cloning Fast Cloning Choose Restriction...restriction cloning is still routinely performed by many labs, a variety of other cloning techniques with...extension PCR. Although each type of cloning has its advantages, most scientists still encounter several...the multi-cloning site, and the reverse primer is located in the 5’ end of the multi-cloning site. The...ve confirmed that the PCR was successful, mix together the vector and insert PCR products with DpnI, and...

  3. Plasmids 101: Colony PCR

    Type
    Blog Post
    Published
    May 12, 2016, 2:30 p.m.
    ...colony PCR.   Designing colony PCR primers The first and perhaps most important step to colony PCR is designing...   PCR set-up Setting up colony PCR reactions is nearly identical to preparing a standard PCR reaction... few positive clones, the last step is to mini-prep these clones and submit the plasmids for Sanger sequencing... between the plasmid and insert DNA. Colony PCR will greatly reduce the number of clones you’ll need to...colony PCR can accomplish the same thing in less time and for less money. The key steps to colony PCR are...kind of test, with a positive clone amplifying a product and a negative clone resulting in no product. Additionally...insert site. A positive clone will produce a larger size product then a negative clone without the insert....

  4. Plasmids 101: TOPO Cloning

    Type
    Blog Post
    Published
    Oct. 27, 2016, 2:30 p.m.
    ...Topoisomerase based cloning (TOPO cloning) is a DNA cloning method that does not use restriction enzymes...TOPO-TA) cloning; however, the TOPO cloning technique has also be adapted for blunt end cloning. So how...using your favorite PCR protocol.  2. Set Up TOPO Cloning Reaction: Mix together the PCR product and TOPO...How to Verify Your Plasmid Resources on Addgene.org Find More Plasmid Cloning Protocols Catch Up On ...does TOPO cloning work? As illustrated in the Figure below, the “A” overhang on the blue PCR product insert...positive clones. Once you introduce your 3’-end “A” overhang insert, the magic of TOPO cloning happens....break down the steps needed for TOPO cloning: 1. Create Your PCR Product: Design standard primers (no ...

  5. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    Published
    Oct. 18, 2018, 12:37 p.m.
    ... posts on plasmid cloning Resources on Addgene.org Read our protocols for plasmid cloning Find our molecular...to amplify by PCR, so we usually add DMSO (3%) and Betaine (1 M) to the PCR mix. When PCR is not possible...sequences. Multiple plasmids can be included in a single PCR, for example for subcloning genes from different...from the MRC Laboratory of Molecular Biology. Plasmid cloning is an essential part of any molecular biology...majority of current cloning techniques involve the assembly of a circular plasmid in vitro, before transforming... bacterial in vivo cloning is now able to come of age. The in vivo assembly cloning protocol In Vivo Assembly...matter if the DNA is PCR amplified, restriction digested or even a synthesized gene. PCR is the primary method...

  6. Plasmids 101: 5 factors to help you choose the right cloning method

    Type
    Blog Post
    Published
    Aug. 21, 2018, 12:31 p.m.
    ... Read other Plasmids 101 blog posts New to plasmids? Read our article on What is a Plasmid? Download the... the Plasmids 101 eBook Resources on Addgene.org0 Check out protocols for plasmid cloning Find vector... thing since CRISPR; you are ready to clone! But...how? Cloning is no longer limited to restriction enzymes...might consider Gibson cloning. In this technique, you first perform a round of PCR to generate DNA fragments...transform the PCR products into E. coli. Ligases within the E. coli then fuse together the PCR products via...over from the PCR process (the precise mechanism isn’t well understood). Finally, TOPO cloning is another...limited - what cloning approach should you use if the budget is particularly tight? Most cloning processes ...

  7. Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

    Type
    Blog Post
    Published
    Sept. 26, 2024, 1:15 p.m.
    ...If cloning methods had personalities, SLIC (sequence- and ligation-independent cloning) would be a true...starting point: Ligation-independent cloning Ligation-independent cloning (LIC) was first developed in the...traditional restriction enzyme cloning uses short sticky ends to insert genes into plasmid backbones, base pairing...strong enough to hold a plasmid together through transformation, so the cloned vector must be ligated....limited to specially designed plasmids. Sequence- and ligation-independent cloning: A SLIC makeover In 2007...hold the plasmid together, even if there are gaps or flaps remaining. This incomplete plasmid looks like...Schematic of SLIC. To start the SLIC cloning process, a gene of interest is PCR amplified to add 5′ and 3′ homology...

  8. Plasmids 101: Screening Strategies Used in Plasmid Cloning

    Type
    Blog Post
    Published
    Sept. 15, 2020, 1:15 p.m.
    ...should expect a PCR product if the plasmid contains the insert and no PCR product if the plasmid does not contain...’re cloning a plasmid, you’ll need a way to find the needle in the haystack: the one perfect clone that...correct clone. Additional resources on the Addgene blog Get all the plasmid basics in our Plasmids 101 ... to use positive and negative selection in plasmid cloning.  But oftentimes, a selection strategy is ...find the desired clone. Do your selection, then screen Why add a screen to your cloning strategy? A screen...once in the plasmid backbone and another enzyme that cuts within the insert. If the plasmid contains the...our restriction analysis blog post. Colony PCR Colony PCR can detect the presence or absence of DNA using...

  9. Troubleshooting Your Plasmid Cloning Experiment

    Type
    Blog Post
    Published
    Sept. 24, 2019, 12:55 p.m.
    ...Browse our Plasmids 101 blog posts Read blog posts on plasmid cloning Read about different plasmid elements...development scientist at Addgene. Cloning can be quite an arduous process. The PCR could fail to produce a product... the correct plasmid. There’s a lot that can go wrong! With all the steps in the cloning process, there...troubleshoot the cloning experiment. Here are some tips that will help you with your cloning project, and ...can assemble the different parts of a plasmid based on the cloning strategy you followed. While most of..., and try a different cloning strategy. Once you've identified potential clones after the screening, sequence...due to a PCR mistake or a mistake by the DNA analysis software. Now that you've read our plasmid troubleshooting...

  10. Site Directed Mutagenesis by PCR

    Type
    Blog Post
    Published
    Aug. 2, 2016, 2:30 p.m.
    ...mutagenesis of Plasmids. A) Generation of mutagenized plasmids. PCR primers (green) amplify the plasmid template...with the nuclease-resistant nicked plasmid (the PCR product). Plasmids are isolated from the resulting colonies...DpnI) to eliminate the parent plasmid from the PCR products, the plasmid template should be isolated from...Blog Brush up on Your Cloning Techniques Learn More about Plasmids in Our Plasmids 101 Series Use CRISPR...can be introduced to plasmids using primers (with the desired mutation) in a PCR protocol that amplifies... mutagenized plasmid. Although it is commonly believed that the final products of the PCR reaction are...the final circular plasmid product pictured here. B) Screening of recovered plasmids for the mutation of...
Showing: 1 - 10 of 79 results