![]() Then we added a second copy of pBR322 into one of the EcoRI sites to create a molecule with two copies of pBR322 and one copy of pBR322+ (Hybrid–2+1). We saved this molecule with the two directly repeated plasmids “ Hybrid–1+1”. ![]() We then joined a copy of both plasmids by selecting the unique EcoRI site in pBR322, selecting Edit | Copy, then selecting the EcoRI site in pBR322+ and choosing Edit | Paste. We used pBR322 and introduced a fifth FspI site by changing the T at 2788 to a C, creating pBR322+. You can simulate this in MacVector by creating concatenated fake plasmids counting the two variant plasmids. You might come across this type of scenario if you have been making site-specific mutations and introducing new restriction sites into a vector where only some of the resulting plasmids might have acquired the extra site. Getting Started with MacVector: An overview of primer design workflows in MacVector.We had a recent support call this week from somebody who believed from their agarose gels that they had a mixed population of plasmids from an experiment and wanted to document and determine the banding pattern using MacVector’s agarose gel simulation.Melissa Caimano on HOW DO I video guides to common molecular biology workflows.admin on HOW DO I video guides to common molecular biology workflows.mariam abdelmalak on Major release details – Summary.Brian on Designing primers and documenting In-Fusion Cloning with MacVector.Chris on Designing primers and documenting In-Fusion Cloning with MacVector.MacVectorTip: displaying CRISPR PAM Sites on a sequence.MacVectorTip: Sign up for an NCBI API key to speed up BLAST results.MacVectorTip: Designing Primers for Gibson Assembly.MacVectorTip: Simulating mixed plasmid populations in agarose gels.MacVectorTip: How to find Restriction Enzymes that only cut outside of a specific region.The window that opens contains a COPY of the starting sequence – now click on the Add Seqs button and select all the ABI/.ab1/SCF chromatogram files from your sequencing project directory to import them into the window In any event, open that sequence first, then choose Analyze | Align To Reference. It can even be the entire genome of an organism to which you want to align chromatograms from sequencing runs of mRNA or cDNA clones. Otherwise, you would normally start with a Reference sequence – it might be the predicted sequence of the PCR fragment you cloned, the starting sequence for a mutagenesis experiment or even a related sequence from another organism. I’m not going to discuss that here but there is a tutorial that you can download from this link. Admittedly, that is not always the case – if you are trying to determine the sequence of an unknown piece of DNA, then you need to use the add-on Assembler module. Typically, if you are aligning chromatograms it is because you are re-sequencing something. None of these algorithms will “flip” the chromatograms when required, but the strategies described below will do that. Note that you should NOT use the Multiple Sequence Alignment ( ClustalW, Muscle or T-Coffee) interface unless you are truly looking for the evolutionary relationships between the sequences and know that they are already all in the correct orientation. One common request we get is “I want to see my chromatograms/traces aligned so that I can edit the alignments”. The blog link discusses the 6 main alignment algorithms in MacVector and how to decide which is the most appropriate for accomplishing different tasks. I blogged about this a few years ago, but its something that still comes up on a regular basis.
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