Worksheet 2 La Trobe University MED 3LAB

Worksheet 2: Attempt review My LMS Subjects / 2021-PSB-MED3LAB(SP-T2-FT) / Assessment / Worksheet 2 Started on Saturday, 12 June 2021, 12:55 AM State Finished Completed on Tuesday, 15 June 2021, 10:59 AM Time taken 3 days 10 hours Grade 24.75 out of 25.50 (97%) Information Worksheet - Constructing the pQE30-GFP expression vector In this exercise, you relate both the theoretical and practical aspects of the protocols used for restriction enzyme digestion, DNA purification, gel electrophoretic analysis and ligation of the pQE30 and GFP cDNA. Please read and follow any instructions carefully as these questions will be automatically graded. 8/3/2021 Worksheet 2: Attempt review Question 1 Correct Mark 1.50 out of 1.50 PART A: Restriction Enzyme Digestion & Purification of Digested DNA The restriction endonucleases used in this practical are purchased from a commercial company. These are supplied at 10 U/μl in 50% (v/v) glycerol. Based on the protocols used in Laboratory Module 2: The digestion reaction contained a total of 40  U of restriction enzyme The final concentration of glycerol in the digestion reaction was 8  % TIP: Be sure to understand what 1 U (unit) of restriction enzyme means in answering this question. Note. Your answers should be to one decimal place only. Do not include units as a part of your answer. There was a total of 4 μl of restriction endonucleases used in the 5 μl of BamHI/HindIII premix. Each 5 μl aliquot contained 2 μl of each restriction enzyme plus 0.5 μl of 10 x Buffer E and 0.5 μl of water. As each μl of RE provides 10 U of enzyme, there is 4 μl x 10 U = 40 U of restriction enzyme in the digestion reaction. --- The restriction endonucleases are stored in 50% (v/v) glycerol (C1) Volume of RE used (V1) = 4 μl Final reaction volume (V2) = 25 μl Therefore, the final concentration of glycerol (C2) = 4 ul/25 ul x 50% = 8% 8/3/2021 Worksheet 2: Attempt review Question 2 Correct Mark 1.00 out of 1.00 Given the total units of BamHI included in the pQE30 digestion reaction and the specified incubation time, what is the theoretical amount of DNA that could be digested under these conditions? Select one: 1 μg 2 μg 4 μg 10 μg 20 μg 25 μg 40 μg  50 μg Your answer is correct. As 2 μl of BamHI (20 units) was used for the digestion of the pQE30 vector, which was performed for 2 h, the theoretical amount of DNA that could be cut was = 20 μg/h x 2 h = 40 μg of DNA can be theoretically cut. Definition: One unit (U) of restriction endonuclease activity is defined as the amount of enzyme required to produce a complete digest of 1 µg of substrate DNA in 1 h (in a total reaction volume of 50 µl) under optimal assay conditions for the restriction endonuclease. Note that while we may have used a 25 µl reaction volume, this may not be optimal but does not halve the restriction endonuclease activity. The correct answer is: 40 μg 8/3/2021 Worksheet 2: Attempt review Question 3 Correct Mark 1.50 out of 1.50 In standard restriction enzyme digestion reactions, manufacturers generally recommend that the final glycerol concentration does not exceed  %. Based on these guidelines, the digestion reaction described in laboratory module 2 is  designed. It is  that ‘STAR ACTIVITY’ would be observed in this reaction. TIP: Be sure to look up the Optimising RE reactions web link and the embedded link to Star Activity. 5 not optimally possible Your answer is correct. It is recommended that the volume of restriction enzymes used in a digestion reaction does not exceed 10%. As the enzymes are stored in 50% (v/v) glycerol, this means that the final glycerol concentration in the should bot exceed 1/10 x 50% (v/v) glycerol = 5% (v/v) glycerol As 8% (v/v) glycerol was used in this experiment, this is poorly designed and star activity is possible. The correct answer is: In standard restriction enzyme digestion reactions, manufacturers generally recommend that the final glycerol concentration does not exceed [5]%. Based on these guidelines, the digestion reaction described in laboratory 8/3/2021 Worksheet 2: Attempt review Question 4 Correct Mark 1.00 out of 1.00 module 2 is [not optimally] designed. It is [possible] that ‘STAR ACTIVITY’ would be observed in this reaction. TIP: Be sure to look up the Optimising RE reactions web link and the embedded link to Star Activity. The BamHI and HindIII restriction enzymes used in this practical were purchased from Promega and used in the recommended Buffer ‘E’. If Buffer H was used instead, what % activity would you expect each of the enzymes to have? BamHI activity in Buffer H = 50  % HindIII activity in Buffer H =