I want to have 301 copies/ul for my downstream experiments. The sample I am using is a purified plasmid, of which I know the concentration (ng/ul), volume (ul), and molarity(g/mol). How can I get my desired DNA copies?
Creating desired DNA copies typically involves a process called DNA amplification, commonly done through polymerase chain reaction (PCR) in molecular biology labs. Here's a simplified overview of how you might go about it:
Design Primers: Primers are short DNA sequences that bind to the target DNA you want to copy. You need two primers, one for each end of the sequence you want to amplify. These primers are designed to match specific regions of the target DNA.
PCR Setup: Prepare a PCR reaction mixture containing the DNA sample you want to amplify, primers, DNA polymerase enzyme, nucleotides (the building blocks of DNA), and buffer solution. This mixture is then placed in a thermal cycler, a machine that can precisely control temperature changes.
PCR Cycling: The PCR machine heats and cools the reaction mixture in cycles. Each cycle typically consists of three steps:Denaturation: The reaction mixture is heated to around 95°C, causing the double-stranded DNA to separate into single strands. Annealing: The temperature is lowered to around 50-65°C, allowing the primers to bind (anneal) to their complementary sequences on the target DNA. Extension: The temperature is raised to around 72°C, and the DNA polymerase enzyme extends the primers by adding nucleotides, creating new DNA strands complementary to the target sequence.
Multiple Cycles: The cycling process is repeated typically 20-40 times, resulting in exponential amplification of the target DNA sequence. After each cycle, the number of DNA copies doubles.
Analysis: Once the PCR is complete, you can analyze the amplified DNA using various techniques such as gel electrophoresis or sequencing to verify the presence and quality of the desired DNA copies.
This process allows you to generate millions to billions of copies of a specific DNA sequence from a tiny amount of starting material, enabling various applications such as genetic testing, sequencing, cloning, and gene expression analysis.
There are probably some online calculators you can use but the process is not that complicated.
What you need to do is calculate your molar concentration (mole/L) based of your molecular weight and ng/µl concentration. Once you have this information you can convert mole to 6.23X1023 molecules giving you the number of molecules per volume you're looking for.