Help with part 2 please! (Perform the necessary calculations for setting up the

Question

Help with part 2 please! (Perform the necessary calculations for setting up the ligation reaction using the information provided.

1. Perform the necessary calculations for setting up your samples to run on a gel in part II of the protocol this week. Necessary information for the calculations: The samples that you need to load are: 1) uncut pUdC 2) pUC/EcoRI digest 3) VEcoRl digest The final volume of each sample that you will load should be 20 The uncut pUC is at a concentration of 0.1 mg/ml, you need to load 0.1 g of pUC. Load approximately 0.1 g of each digest reaction so you will need to first figure out the concentration of DNA that is contained in the digest reaction using your calculations and protocol from last week. All samples need 1x loading dye -it is supplied as a 6x stock. Use TE to balance the volume for each sample. 2. Perform the necessary calculations for setting up the ligation reactions using the information provided in part IV of the lab protocol for this week. 3. There's a lot going on this week in lab...show me that you understand how everything fits together by drawing a simple flow chart of the activities. It doesn't need to be a work of art but should be legible and easy to follow.

Explanation / Answer

2-

given that we need to 0.1 micrograms and the stock concentration is 0.1mg/ml which is equal to 0.1microgram/microlitre.

1 mg = 1000 microgram

1ml = 1000 microlitre.

So we need to take 1 microlitre from the stock solution to load on the gel.

0.1 microgram = 100ng.

Total pUC in 20 microliter reaction is 100ng.

So the concentration of pUC is 5ng/microlitre.

For ligation, we need to use 2 microlitres digested pUC. So the amount of pUC is 10ng (5ng/microlitre).

Since nothing is given about the concentration of Lambda.EcoRI digest so I will take its concentration same as that of pUC.

SO the concentration of Lambda.EcoRI digest is 5ng/microlitre and we need to take 10microlitre of the digest. So the amount is 5*10 = 50ng.

pUC = 2 microlitre

Lambda.EcoRI 10 microlitre.

10 X T4 DNA ligase buffer = 2 microlitres.

T4 DNA ligase = 1 microlitre.

Sterile water = 5 microlitre

Write me back if you have any doubt.

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