I am supposed to make different ligations.
These are the stocks I am given:
Cut pET41a(+) DNA: 25 ng/ul
egfp insert DNA: 7 ng/ul
Uncut pET-41a(+)/EGFP recombinant plasmid DNA: 25 ng/ul
This is the ligation I am supposed to make:
Ligation #1: 1:1 molar ratio pET-41a(+) vector: egfp insert
-50 ng NcoI/NotI cut pET-41a(+) DNA _______ ul
-7 ng egfp insert DNA _____?l
-sterile dH2O _____?l
-10x ligase buffer _____ ul (final concentration will be 1X)
-DNA ligase: 1 ul
The final volume for the ligation is 20ul.
I am trying to find out how ul of the stock I am supposed to add for the cut and I am having trouble. I am using the formula C1V1=C2V2. When I solve I keep getting 40 ul but i know that's wrong.
Well, well, well, it seems like someone's having trouble with their ligation math. Don't worry, my friend, the Clown Bot is here to help you out and add some laughs along the way!
Let's break it down step by step. We know that you need a 1:1 molar ratio of pET-41a(+) vector to egfp insert. The final volume of your ligation is 20 ul. So, we'll use the equation C1V1 = C2V2 to figure out the volume of each component.
First, let's solve for the volume of the pET-41a(+) vector:
C1V1 = C2V2
(25 ng/ul) V1 = (50 ng) (20 ul)
Now, let's plug in the values and solve:
(25 ng/ul) V1 = (50 ng) (20 ul)
V1 = (50 ng) (20 ul) / (25 ng/ul)
V1 = 40 ul
So, according to my calculations (which are always correct because I'm a super intelligent Clown Bot), you should add 40 ul of the cut pET-41a(+) DNA stock to your ligation.
Remember, science can be tricky, but never forget to add a little humor along the way. Good luck with your ligation, my friend!
To determine the volume of the stock you need to add for the cut pET-41a(+) DNA, you can use the formula C1V1 = C2V2, where C1 is the initial concentration of the stock, V1 is the unknown volume you need to find, C2 is the final concentration you desire, and V2 is the final volume.
In this case, you know that the final volume of the ligation is 20 µl, and you want a 1:1 molar ratio between the pET-41a(+) vector and the egfp insert DNA. Therefore, the concentration of the pET-41a(+) vector and the egfp insert DNA should be the same.
Let's assume the required final concentration is X ng/µl. Therefore, using the formula:
(25 ng/µl)V1 = (X ng/µl)(20 µl)
V1 = (X ng/µl)(20 µl) / (25 ng/µl)
V1 = (20/25)X µl
From this, we can see that V1 is equal to (20/25)X µl. Therefore, the volume of the stock to add for the cut pET-41a(+) DNA will be (20/25) times the final concentration (X) you desire.
For example, if you want a final concentration of 50 ng/µl, the volume of the stock to add for the cut pET-41a(+) DNA would be:
V1 = (20/25)(50 µl) = 40 µl, which is the answer you obtained.
So, it seems like your calculation is correct, and you should add 40 µl of the stock for the cut pET-41a(+) DNA.
To calculate the volume of the stock solution you need to add for the cut pET-41a(+) DNA, we can use the formula C1V1 = C2V2, where C1 and V1 represent the concentration and volume of the stock solution, and C2 and V2 represent the desired concentration and volume.
Given:
C1 = 25 ng/µl
V1 = ?
C2 = 50 ng/µl (assuming a 1:1 molar ratio)
V2 = volume of cut pET-41a(+) DNA needed (to be determined)
First, let's convert the concentrations to ng/µl to maintain consistency:
C1 = 25 ng/µl
C2 = 50 ng/µl
Now, we can rearrange the formula C1V1 = C2V2 to solve for V1:
V1 = (C2V2) / C1
Substituting the values:
V1 = (50 ng/µl * V2) / 25 ng/µl
Simplifying:
V1 = 2 * V2
Now, we know that the final volume for the ligation is 20 µl. The total volume of cut pET-41a(+) DNA and egfp insert DNA should equal this final volume. Given that the volume of the egfp insert DNA is not specified, we can calculate it as follows:
Volume of egfp insert DNA = final volume - volume of cut pET-41a(+) DNA
Volume of egfp insert DNA = 20 µl - V1
Since we are assuming a 1:1 molar ratio, the volume of egfp insert DNA and cut pET-41a(+) DNA are the same.
Thus, the final equation becomes:
V1 + V1 + 20 µl - V1 = 20 µl
Simplifying the equation:
V1 = 40 µl / 2
V1 = 20 µl
Therefore, you need to add 20 µl of the stock cut pET-41a(+) DNA solution to achieve the desired concentration and volume for Ligation #1.