Worksheet 1 La Trobe University MED 3LAB

Worksheet 1: Attempt review My LMS Subjects / 2021-PSB-MED3LAB(SP-T2-FT) / Assessment / Worksheet 1 Started on Friday, 4 June 2021, 6:00 PM State Finished Completed on Sunday, 6 June 2021, 5:33 PM Time taken 1 day 23 hours Grade 12.00 out of 15.00 (80%) Information Worksheet 1 One of your first tasks in the practicals is to perform a PCR to obtain large quantities of the GFP cDNA from the pE-GFP vector. Following digestion with appropriate restriction enzymes, this DNA will be ligated in the correct reading frame into the pQE30 vector, resulting in the production of a GFP-hexahistidine tagged gene fusion. In order to understand this process fully, it is essential for you to gain some experience in identifying ORFs and restriction enzyme sites. It is also important that you understand some of the factors involved when designing oligonucleotides to act as PCR primers. These considerations will be addressed in this activity. The activity is divided into 3 Parts and are designed to complete in order. For each part, you will be required to download a PowerPoint slide and use the instructions on the slide to complete the exercises. After you complete each section, you should answer the associated questions within the quiz. Note: As this is an interactive PowerPoint slide, it is essential that you do not change the sizes of text boxes or font sizes when working through each activity. 8/3/2021 Worksheet 1: Attempt review Information Question 1 Correct Mark 2.00 out of 2.00 PART A: THE pQE30 VECTOR This activity should be used for examining the features of the pQE30 vector and establishing what the vector will theoretically look like following digestion with restriction enzymes. To get started, download Part A of the activity here. You should save a copy of this PowerPoint file onto your own computer and work through the activity. Once complete, you can answer the associated quiz questions and upload your activity below. Within the pQE30 vector sequence, you should have identified the region encoding the 6xHis-tag. Write the DNA nucleotide sequence that encodes for this tag here: CATCACCATCACCATCAC  NB. You should include the sequence for the 'coding' strand only (5' →3'). Use capital letters to enter your response. Do not include numbers or spaces as a part of your answer. 8/3/2021 Worksheet 1: Attempt review Question 2 Correct Mark 1.00 out of 1.00 The pQE30 vector contains a number of restriction enzyme sites within its 'Multiple Cloning Site'. Match each of the restriction enzymes below with it's nucleotide recognition site.       BamHI HindIII KpnI PstI SacI SalI Your answer is correct. There are various commercial companies that purify and sell restriction endonucleases. The positions at which restriction enzymes recognise and digest a DNA sequence can be found by looking up the company catalogs. A lot of additional information can also be found at these sites, including: the optimal temperature at which the enzymes work the type of buffer in which optimal digestion can be achieved the concentration that the enzyme should be used whether the enzyme displays 'star activity' whether the enzyme requires additional protein to function well etc In your own time, you should explore some of these sites. One company that sells these products in NEB (New England Biolabs) The correct answer is: → BamHI, → HindIII, → KpnI, → PstI, → SacI, → SalI 8/3/2021 Worksheet 1: Attempt review Question 3 Complete Not graded Information Save your PowerPoint file with the appropriate file name (Worksheet 1_Part A_Student Number) and upload it as an attachment below. Note: There is no requirement for you to add any written work in the accompanying text box. Worksheet 1_Part A_ PART B: Amplification from pEGFPN1 This activity should be used for identifying the open reading frame (ORF) that encodes GFP and identifying where the PCR primers bind the sequence. In completing this activity, you should be able to determine the size and sequence of the PCR product that we are expecting within the lab. To get started, download Part B of the activity here. You should save a copy of this PowerPoint file onto your own computer and work through the activity. Once complete, you can answer the associated quiz questions and upload your activity below. 8/3/2021 Worksheet 1: Attempt review Question 4 Correct Mark 2.00 out of 2.00 Within this exercise, you should have identified where each of the primers bind to the template DNA. You should have also identified the portion of the primer that does not anneal to the template. For each primer, write the short nucleotide sequence that does not anneal to the template DNA during the PCR Forward Primer Overhang: 5' GCGCAGGGATCC  3' Reverse Primer Overhang: 5' CAGGCGAAGCT  3' NB. Write your DNA sequence 5' → 3'. Use capital letters only. Do not include numbers or spaces as a part of your answer. 8/3/2021 Worksheet 1: Attempt review Question 5 Incorrect Mark 0.00 out of 1.00 Not all oligonucleotide primers are designed with 5' overhanging regions that do not anneal to template DNA. What is the purpose of these overhangs in PCR? Select one: They are used to optimise the Tm of a primer They are used to incorporate new features into the final nucleotide sequence They are used to facilitate DNA polymerase binding during the PCR reaction All of the above  Your answer is incorrect. Choice 1 - The 5' overhang is NOT typically used to increase the Tm of a primer. Additional nucleotides are frequently added to increase the Tm of an oligonucleotide primer. As the Tm or annealing temperature of the primer is most critical in the first cycle of the PCR, annealing temperatures should be modified with the template in mind. Thus, when adding nucleotides for the purpose of increasing annealing temperature, these would typically be added to the 3' end of a primer. Remember, 5' overhangs are not engineered to bind to the original template. Choice 2 - A 5' overhang CAN be used to incorporate NEW features into a nucleotide sequence. Additional nucleotides are frequently added to the 5' end of oligonucleotides to incorporate RE sites for cloning purposes. They can also be added for other purposes such as incorporating sequences that encode specific amino acids at the N- or C- terminus of a protein sequence. For example, this could be to encode an epitope tag or protease cleavage site. Choice 3 - A 5' overhang is NOT needed to facilitate DNA polymerase binding in PCR. PCR reactions can proceed perfectly well without the addition of nucleotides that do not bind to the template DNA. Choice 4 - This is NOT the correct answer. From all the options provided, only Choice 2 is relevant. The correct answer is: They are used to incorporate new features into the final nucleotide sequ