You wish to perform a restriction digest in a 20uL volume. Your DNA is 0.20 ug/uL and you wish to digest 1.0 ug. Your restriction enzyme is provided as 0.2 U/uL. Fill in the appropriate volumes: H2O, 10Xbuffer, DNA, and restriction enzyme.
Please show work. I'm more interested in how to get the answer. Thanks.
To calculate the volumes needed for the restriction digest, you need to consider the concentrations of the DNA and the restriction enzyme, as well as the desired amount of DNA to be digested.
Let's break down the calculation step by step:
Step 1: Calculate the volume of DNA needed based on the desired amount to digest.
Given:
DNA concentration = 0.20 ug/uL
Desired amount to digest = 1.0 ug
To calculate the volume of DNA needed, use the formula:
Volume of DNA (uL) = Desired amount to digest (ug) / DNA concentration (ug/uL)
Substituting the given values:
Volume of DNA (uL) = 1.0 ug / 0.20 ug/uL
Volume of DNA (uL) = 5.0 uL
Therefore, you would need 5.0 uL of DNA.
Step 2: Calculate the volume of the restriction enzyme needed.
Given:
Restriction enzyme concentration = 0.2 U/uL
To calculate the volume of the restriction enzyme needed, use the formula:
Volume of restriction enzyme (uL) = Desired amount of enzyme (U) / Enzyme concentration (U/uL)
In this case, the desired amount of enzyme is not given, so we will assume it is 1.0 U (a common amount for restriction digests).
Volume of restriction enzyme (uL) = 1.0 U / 0.2 U/uL
Volume of restriction enzyme (uL) = 5.0 uL
Therefore, you would need 5.0 uL of the restriction enzyme.
Step 3: Calculate the volume of the 10X buffer needed.
In a restriction digest, the volume of the 10X buffer is typically one-tenth of the total reaction volume. In this case, the total volume is 20 uL.
Volume of 10X buffer (uL) = Total volume (uL) / 10
Volume of 10X buffer (uL) = 20 uL / 10
Volume of 10X buffer (uL) = 2.0 uL
Therefore, you would need 2.0 uL of the 10X buffer.
Step 4: Calculate the volume of H2O needed to reach the desired total volume.
Given:
Total volume = 20 uL
To calculate the volume of H2O, you subtract the volumes of DNA, restriction enzyme, and 10X buffer from the total volume:
Volume of H2O (uL) = Total volume (uL) - Volume of DNA (uL) - Volume of restriction enzyme (uL) - Volume of 10X buffer (uL)
Volume of H2O (uL) = 20 uL - 5.0 uL - 5.0 uL - 2.0 uL
Volume of H2O (uL) = 8.0 uL
Therefore, you would need 8.0 uL of H2O.
In summary, the volumes needed for the restriction digest in a 20 uL total volume are as follows:
- H2O: 8.0 uL
- 10X buffer: 2.0 uL
- DNA: 5.0 uL
- Restriction enzyme: 5.0 uL