enzymes being used here are at a concentration of 10 U/uL. For double digests, give the appropriate volume for each enzyme and decide which buffer to use. (HINT: look up double digest conditions on the NEB website.) You should always calculate all volumes in advance to ensure the correct working concentrations and so that you can prepare the digests as efficiently as possible. It is a good idea to check off each component as it is added to the microfuge tube. Complete the table below, including the volume of DNA sample(s) you will need for 1 ug of the DNA (from two different methods in Experiment # 3) based on the concentration(s) you determined from the OD250 value, which you will restriction digest with each of the restriction enzymes singly and in double digests in Part A. For evample the volume of DNA sample that contains 1 ug DNA for DNA sample from Part A
enzymes being used here are at a concentration of 10 U/uL. For double digests, give the appropriate volume for each enzyme and decide which buffer to use. (HINT: look up double digest conditions on the NEB website.) You should always calculate all volumes in advance to ensure the correct working concentrations and so that you can prepare the digests as efficiently as possible. It is a good idea to check off each component as it is added to the microfuge tube. Complete the table below, including the volume of DNA sample(s) you will need for 1 ug of the DNA (from two different methods in Experiment # 3) based on the concentration(s) you determined from the OD250 value, which you will restriction digest with each of the restriction enzymes singly and in double digests in Part A. For evample the volume of DNA sample that contains 1 ug DNA for DNA sample from Part A
Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
Chapter1: The Human Body: An Orientation
Section: Chapter Questions
Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...
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Transcribed Image Text:Calculate these for 1 ug of plasmid DNA in a final total volume of 20 ul. All
enzymes being used here are at a concentration of 10 U/µL. For double digests, give the
appropriate volume for each enzyme and decide which buffer to use. (HINT: look up double
digest conditions on the NEB website.) You should always calculate all volumes in advance to
ensure the correct working concentrations and so that you can prepare the digests as efficiently
as possible. It is a good idea to check off each component as it is added to the microfuge tube.
Complete the table below, including the volume of DNA sample(s) you will need for 1 pg of the
DNA (from two different methods in Experiment #3) based on the concentration(s) you
determined from the OD260 value, which you will restriction digest with each of the restriction
enzymes singly and in double digests in Part A.
For example, the volume of DNA sample that contains 1 µg DNA for DNA sample from Part A,
Experiment 3: OD260 = 0.024, dilution 500x
Concentration of DNA (ug/mL) = Az60 x dilution factor x 50 ug/mL = 600 µg/mL
Vol DNA per 1 pg = 1000 µL/600 µg = 1.67 µL
Table 4-1: Recipe for a restriction enzyme digestion reaction
Reagent
Stock Concentration Volume for the Digestion Reaction (uL)
Water
N/A
ul
Buffer
10x
2 µl
DNA
5_ul (from Lab# 3)
uL
Restriction Enzyme(s)
10 Units/ul
1 μL
Total
N/A
20 µl
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