Question

PLEASE SHOW ALL WORK IF POSSIBLE! THANK YOU IN ADVANCE.

1) SOC media is 2% tryptone, 0.5% yeast extract, 10 mM NaCL (F.W. 58), 2.5 mM KCl (F.W. 74.5), 10 mM MgSO₄ (F.W. 246.5), 10 mM MgCl₂ (F.W. 203.3), 20 mM glucose (F.W. 180.16). Determine the amounts of each to make 500 mL of SOC.

2) Determine the number of moles in 0.05 μg of 300 bp insert DNA. There are 660 g/mol-bp. What is the molar ration of insert to vector if you have 10 ng of vector that is 2686 bp?

9) Transformation efficiency is defined as the number of transformants per μg of DNA. pUC19 DNA (10 μL of 0.005 mg/ml solution is added to 40 μL of E.coli cells in suspension. The solution is put into an electroporation cuvet and shocked. 450 μL of SOC media is quickly added and the cells are incubated at 37 degrees celscius for 60 minutes. Then, 10 μL of this solution is pipetted onto an LB/AMP plate and spread out. Following an overnight incubation at 37 degrees celscius, 576 transformant colonies were found growing on the plate.

a. How much DNA was in teh electroporation cuvet?
b. How much DNA was int eh 10 μL aliquot that was plated?
c. What is the transformation efficiency of this experiment?

10) Set up a restriction digest of 5 μg of pBR325 DNA is enough HindIII restriction enzyme to fully digest the DNA (in one hour) in a final concentration of 1X restriction digest buffer (with water). The soluitons you have to start with are: water, HindIII stock at 10 units/μL, 0.5 mg/mL pBR325, and 10 x stock restriction digest buffer. What volumes of each of these components do you need to add for a 40 μL restriction digest?

Answer 1

1) 5ml of tryptone and 2.5 ml of yeast extract

290 mg of NaCl

0.093 mg of KCl

1.23 mg of MgSO4

1.016 mg of MgCl2

3.60 mg of Glucose