MARK ROBERT MAGSINO 2018-00449 CMB 3101-4

Experiment 5: Primer Design and PCR

Objectives:
• Be familiar with the steps of Polymerase Chain Reaction (PCR)
• Understand the importance of each PCR component
• Be familiar with the rules of primer design
• To do basic primer design using NCBI database

Watch the following videos about PCR:

Introduction to PCR: https://youtu.be/Nl6eLez3CNI

Basics of PCR: https://youtu.be/mOKb0Pd_Rac

Principles of PCR: https://youtu.be/VD5qEVTsjTc

How to Set Up a PCR Reaction: https://youtu.be/NYlT3f-MZ5o

Watch the following videos about Primer Design:

Tips for design PCR Primer: https://youtu.be/OcN6mML3DGI

PCR Primer Design Overview: https://youtu.be/ml0kxx2OJ1A

Watch Interactive simulation:

(https://www.labxchange.org/library/items/lb:LabXchange:2960e770:lx_simulation:1)
MARK ROBERT MAGSINO 2018-00449 CMB 3101-4

Watch the following videos about Restriction Digest:

Restriction Digestion of DNA: https://youtu.be/GsWo8dCivWs

How Do I Set-up a Restriction Enzyme Digest: https://youtu.be/_HKUiyVhBpo

Resource Database:

National Center for Biotechnology Information Gene Page: https://www.ncbi.nlm.nih.gov/gene/

National Center for Biotechnology Information Main Page: https://www.ncbi.nlm.nih.gov

National Center for Biotechnology Information Primer-BLAST Page:

https://www.ncbi.nlm.nih.gov/tools/primer-blast/

Example Companies focused on Next Generation Sequencing:

Promega NGS: https://worldwide.promega.com/products/sequencing/

Thermo Fisher Scientific NGS: https://www.thermofisher.com/ph/en/home/life-

science/sequencing/next-generation-sequencing.html

Bio-Rad NGS: https://www.bio-rad.com/en-us/category/next-generation-sequencing?ID=PZ1788TU86LJ

New England BioLabs NGS: https://international.neb.com/applications/ngs-sample-prep-and-target-

enrichment
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Experiment:

1st Activity:
Primer Design & Restriction Digest

This is the whole sequence of the K. pneumoniae lac operon (https://www.ncbi.nlm.nih.gov/nuccore/M11441.1),

highlighted is the sequence for the lac repressor lacI. With the knowledge you have learned from the videos

above, do the following and show your work on the calculations:

1. Design a forward and reverse primer for the lac repressor (lacI) [Highlighted below]. Do not use the first

18 to 30 bp from both ends of the highlighted sequence.

2. What will be your temperature settings for PCR? Given that your cycles are the standard 25-30 cycles and

using the 4+2 rule.

mT = 4(G+C) + 2(A+T)

Forward primer Reverse primer

C-7 G-4 A-3 T-6 = 20 C-4 G-7 A-3 T-6 = 20

4(4+7) + 2(3+6) = 62 degrees Celsius 4(7+4) + 2(3+6) = 62 degrees Celsius

3. You want to insert a sequence in the lacI gene to alter its function, what restriction enzyme(s) will you

use? Indicate 1 or 2 restriction enzyme(s) that you will use on the gene to show the cut.

1 aagcttctcc accagcgctt tcagctgcgc cagggtctgc tgccggtcct ccgcggcggc

61 gacaggccag gccgactggc gcacgatgag ccgggtcggc agcagctcgc ggatccgcag
121 ctgcggggcc gccatcaggg caatcagccg ctccaccgcc cttttgccca acagatcgaa
181 gtcctgcgcc accgtggtga gcggcggctg gaagtaaagg ctgtcggcgg tgtcatcgta
241 gccggtcacc gataccgcct ggctgccgct gcgattgagc tgggccagcg cgctgagcac
301 gccgagcgcc atctgatcgt ttgccaccac tatggcgctg atccgcggct gcaggtggag
361 gagctcgaaa gttttctgcc agccgctggc ggcgctccag tcgccaaaca ccgtagtaga
421 gcgggcaata ttcagactgt gcaacgcctc gcgccagctg gcgagacgca gacgggcgga
481 aaccgaactt tccggtcccg ccagcagacc aaattcgcga tgccccatct cccacaggtg
541 gcgcacgcag gccccgcagc cgtcgcggtg gtcgaagcgc acgcagcaga catcggcctc
601 cggggagaca tcgagaaaca ggcaggccat atccggattg tcttgcacca gccgctcggc
661 agtggcgctc tccagcggca gactgacgat caccccgcga atatgctggg cgcgcagctc
721 gtccagccgg gcctgcagcg cgacaaaatc ggcctgcgcc ggcatcgcta tcgccacttc
781 cagctgatgc aggctggcat ggctctttat cgcggcggcg atctgcgagg gggcgtgcag
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841 cgtcacggag gcggtaatca gcccgatgga gggcgccgct ttgccggcaa gcagctgcgc

901 cgagcggtta ggcacatagt gcagcgcctg catcgcgcgg atcacctgct cgcgggtacg
961 ggctgagacc acctcaggac ggttcagcac ccggcggaga ccgtctgctg ggacacgccc
1021 gcggcgcgcg acatcctcca gggttgcggt acgacgcggc atcttttctc ctcagtaata
1081 acaatgtcgt gcgctaacat tttatgttta acgcgcgaca aaaaacagcg cttcgccccc
1141 gctttcgtga tcgtctgtta atttcgattc ccccgagatt gctggacaaa acaaattacc
1201 cgataacgtt aaccatcctg tttagcgaac gacaatttct gacttaccgg ggtttaatat
1261 gcaaattagc gataccggcc gcagccacac tcctgacttt cacgccgtcc tcgcccgtga
1321 agactggcac aaccagacca ttacccacct taaccgcctg ccagcgcatc ccgttttcgc
1381 cagctggcgc gatgagcttg ccgcccgcga taacctacct tcatcccgcc gccgtcaact
1441 ggacgggagt ggcagttctc ttacgcccgc agcccgtttg ccgtcgatgc gagtggtgac
1501 gcaggatcta ccggactgcc gcggcacgcc tgtgccttcc aactggcaga tggagggcta
1561 tgacgcgccg atctacacca acgtccgcta tcccatcgac accaccccac cgcgggtgcc
1621 ggaggataac ccgaccggct gctactccct gcactttacg gttgaggaca catggcgtga
1681 gaacgggcaa acgcagatta ttttcgatgg cgtcaactcg gcgtttcatc tgtggtgcaa
1741 tggcgtgtgg gtcggctatt cgcaggacag tcgcctgccg gcggcgttcg atctcagccc
1801 cttcctgcgt ccgggcgaca accgcctgtg cgtgatggtc atgcgctgga gcgccggcag
1861 ctggctggaa gaccaggata tgtggcggat gagcggcatt ttccgctcgg tatggctgct
1921 gaataagccg cagcaacggc tatgcgacgt gcagttgacg ccagcccttg acgccctcta
1981 tcgcgacggc actctgcagg tccaggcgac catcgaagcg actgaggcgg cgcttgccgg
2041 gctcagcgtc ggggttagcc tgtggcgcgg cgaggagcag ttcgccgccg ggcggcagcc
2101 gttaggtacc ccgacggtgg atgagcgcgg ccactacgcg gaacgggtcg atttctccct
2161 ggcggtggcg acgccggcgc actggagcgc ggaaaccccg aactgttatc gcgccgtggt
2221 caccctgtgg cgcggcgacg aactgctgga ggccgaagcg tgggacatcg gttttcgccg
2281 catcgagatt gccgatggcc tgctgcgtct caacggtaaa ccgctgctga tccgcggcgt
2341 taaccgccat gagcatcatc atttgcgcgg gcaggtggtc accgaggcgg atatggtgca
2401 ggacattctg ttgatgaagc agaacaactt taacgccgtg cgctgctcgc actatcccaa
2461 cgcgccgcgc tggtatgaac tctgcaaccg ctacggtctg tacgtggtcg atgaagccaa
2521 tattgaaacc cacgggatgg tgccgatga a tcggctgtcc gacgatccgg cgtggctacc
2581 agccttcagc gcccgcgtca cccggatggt acagagcaac cgcaaccatc cgtgcattat
2641 catctggtcg ctgggcaacg agtccggcgg cggcggcaac cacgaagcgc tgtaccactg
2701 gctgaaacgc aacgatccga gccgtccggt gcagtacgag ggcggcggcg cggataccac
2761 cgccaccgat attatctgtc cgatgtacgc ccgcgtcgaa cgcgaccagc cgatcccggc
2821 ggtacccaaa tgggggatca aaaaatggat cagcctgccc ggtgagcaga ggccgctgat
2881 cctttgcgag tacgcccacg cgatgggcaa cagcctcggc aacttcgccg attactggca
2941 ggcctttcgc gagtatccgc ggctgcaggg cgggtttatc tgggactggg ccgaccaggc
3001 gatccgcaaa acctttgccg acggcagcgt cggctgggcc tatggcggcg actttggtga
3061 taagcctaac gatcgccagt tctgtatgaa cggtctggtg tttcccgatc gcacgccgca
3121 tccgtcgctg gtggaggcga agcacgccca gcaatatttt cagttcacgc tgctgtcgac
3181 ctcgccgctg cgggtgcgca tcatcagcga atacctgttc cgcccaaccg ataacgaagt
3241 cgtgcgctgg caggtgcagg cggccggtga acccctgtat cacggcgacc tgaccctggc
3301 gctgccccct gagggcagcg acgagataac gctgctcgat agcctgatcc tgcctgaagg
3361 cgcccgcgcg gtgtggctga cgctggaggt gacccagccc caggcgaccg cctggtcaga
3421 agcggagcac cgcgtcgcct ggcaacagtt tcccctgccc gccccgctcg gctgccggcg
3481 cccaccgtgt ctgccggcgc tcccggatct tatcgtcagc gatgaggtct ggcagatccg
3541 cgccggttcg caatgctgga ccatcgatcg ccggacgggt ctgctgagcc gctggtcggt
3601 tggcggtcag gagcagctgt tgactcccct gcgtgaccag tttattcgcg cgccgctcga
3661 caacgacatc ggggtcagcg aagtagagcg tatcgacccc aacgcctggg tggagcgctg
3721 gagaagcgcc ggcctgtacg atcttgaggc gcactgcgtc cagtgcgatg cgcagcgcct
3781 ggcaaatgaa accctcgtcg actgccgctg gcactacctg cgcggcgaag aggtagtgat
3841 tgtcagccac tggcgcatgc acttcactgc tgacggaacc ctgcggttgg cagtggacgg
3901 cgaacgggcg gaaaccctgc cgccgctgcc gcgggtcggg ctgcacttcc aggtggcgga
3961 tcagcaggcg ccggtgagct ggctgggtct ggggccgcat gagaactacc ccgaccggcg
4021 gagcagcgcc tgcttcgccc gctgggagca gccgctggcg gcgatgacca ccccctacat
4081 cttcccgacg gaaaacggcc tgcgctgtga tacccaggcg ctggactggg ggcgctggca
4141 catcagcggt catttccact tctccgttca gccatggagc acccgtcagc tgatggagac
4201 cgaccactgg cacaagatgc aggccgaaga cggcgtgtgg atcaccctcg acggcctgca
MARK ROBERT MAGSINO 2018-00449 CMB 3101-4

4261 tatgggggtg ggaggcgatg actcctggac ccccagcgtg ctgccgcagt ggctcctgag

4321 ccaaacgcgc tggcagtacg aggtctcatt gcgtagcctt taatccgtgg gggcgacagc
4381 ccccacccca caaacagaat aacaagggat catggtgatg aaattctcag aactggcgcc
4441 acgagaacgg cataatttcg tctatttcct gctgttcttt ttcttttacc atttcattat
4501 gtcggcctac ttcccgtttt ttccggtgtg gctggcggac gttaaccatc taactaaaac
4561 ggaaaccggg atcgttttct cgtctatctc gttattcgcc attatttttc agccggtgtt
4621 cggcctgatg tcggataagc tcggcctgcg caaacatctg ctgtggacca ttacggtact
4681 gttaattctg ttcgcgccat tctttatttt cgttttctcc ccgctgctgc agatgaatat
4741 tatcgctggt tcgctggtag gcgggatcta cctggggatt gttttctcga cggctccggg
4801 cgtcggaagc tt
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Polymerase Chain Reaction

Given the tables below answer the following questions and show your work on the calculations:

For Table 1:

1. In the given table, what components should you mix together to make your master mix?

To obtain a master mix, substances that should be mix together are Buffer to resist changes in pH
when additional substances are added, dNTPs provides nucleotides to the unzipped strand using the
template of a single side, Taq Polymerase is an enzyme that assembles the nucleotides into a
new DNA strand, MgCl2  an essential cofactor that promotes the activity of Taq DNA polymerase which
increases the amplification rate of DNA.

2. Calculate the volumes of each component for 1 reaction.

DF = initial concentration / final concentration

Dillution factor of :
Water = N/A
Buffer = 5/1 =5
MgCl2 = 25 mM/1.5 mM = 16.67
dNTPs = (10x1000)/200=50
Forward Primer = 10/1 = 10
Reverse Primer = 10/1 = 10
Taq polymerase =5/1.25 = 4
DNA template = N/A

Volume for 1 reaction = Single PCR volume / DF

Water = 25-(5+1.5+0.5+2.5+2.5+6.25) = 4.75 ul
Buffer = 25/5=5 ul
MgCl2 = 25/16.67 = 1.5 ul
dNTPs = 25/50 = 0.5 ul
Forward Primer = 25/10=2.5 ul
Reverse Primer = 25/10 = 2.5 ul
Taq polymerase =25/4=6.25 ul
DNA template = N/A

3. Calculate the volumes of each component for 15 reactions.

Volume for 15 reactions
Water = 4.75 ul x 15 = 71.265 ul
Buffer = 5 ul x 15 = 75 ul
MgCl2 = 1.5 ul x 15 = 22.485 ul
dNTPs = 0.5 ul x 15 = 7.5 ul
Forward Primer = 2.5 ul x 15 = 37.5 ul
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Reverse Primer = 2.5 ul x 15 = 37.5 ul

Taq polymerase =6.25 ul x 15 = 93.75 ul
DNA template = N/A

For Table 2:

1. Calculate the volumes of each component for 1 reaction.

2. Calculate the volumes of each component for 25 reactions.

DF = initial concentration / final concentration

DF of :
Taq 2x = 2/1=2
Forward Primer = 10/0.2 = 50
Reverse Primer = 10/0.2 = 50
Template DNA = N/A
Nuclease free water = N/A

Volume for 1 reaction = Single PCR volume/ DF

Volume for 1 reaction Volume for 25 reaction
Taq 2x = 50/2= 25 25 x 25 = 625 ul
Forward Primer = 50/50 = 1 1x25 = 25 ul
Reverse Primer = 50/50 = 1 1x25 = 25 ul
Template DNA = N/A
Nuclease free water = N/A

Answer the following questions in 2-4 sentences:

1. Why should you not add template DNA, DNA Polymerase, and Forward-Reverse

Primers together when making a master mix?

Adding the template DNA, DNA polymerase, forward and reverse primers will start a

reaction in a tube, in which the DNA polymerase will attach to a DNA region and

primers will start annealing (stick) to the template DNA, and will become the starting

point for DNA replication by selecting the region of DNA that will be amplified which is

unpractical and will provide unspecific products when master mix is used in pcr
 MARK ROBERT MAGSINO 2018-00449 CMB 3101-4

reaction, additionally, the resulting mastermix will not be usable for future

experimentation.

2. Case Study: Your principal investigator asked you to design primers for a project, but with

how busy you are with exams, writing reports, and your thesis you rushed the design of the

primers. Without proofreading the primers, your PCR run with the primers gave bad results

and you noticed very high GC content and insufficient quantity of DNA amplified. How can

you troubleshoot this?

PCR products with very high GC content and insufficient quantity of DNA amplified can
be resolved by examining the quantity of DNA input and increasing the amount if
needed, can also be solved by increasing the annealing temperature. For greater result,
annealing temperature can be optimize by using a  thermal gradient or by using DMSO
or another secondary structure destabilizer which can be added to break down the
bonds that join the two strands of the DNA double helix which enables the DNA to
separate into two single strands and to choose DNA polymerases with  high
sensitivity for amplification and If necessary, number of PCR cycles can be increase.

PCR Components
Ingredient Initial Volume for 1 Final reaction Volume for 15 Volume for 16
Concentration reaction reactions reaction (N+1)
15+1
Water N/A 4.75 ul N/A 71.26 ul 76.01 ul
Buffer 5x 5 ul 1X 75 ul 80 ul
MgCl2 25 mM 1.499 ul 1.5 mM 22.485 ul 24 ul
dNTPs 10 mM 0.5 ul 200 uM 7.5 ul 8 ul
Forward Primer 10 uM 2.5 ul 1 uM 37.5 ul 40 ul
Reverse Primer 10 uM 2.5 ul 1 uM 37.5 ul 40 ul
Taq Polymerase 5 units 6.25 ul 1.25 units 93.75 ul 100 ul
DNA Template N/A 2 ul N/A 30 ul 32 ul
Total 25 ul 375 ul 400 ul
Table 1: Taq DNA Polymerase with Standard Buffer
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Component Initial Volume for 1 Final Volume for 25 Volume for

concentration reaction Concentration reactions 26 reaction
(N+1)=25+1
Taq 2X Master Mix 2X 25 ul 1X 625 ul 650 ul
Forward Primer 10 uM 1ul 0.2 uM 25 ul 26 ul
Reverse Primer 10 uM 1ul 0.2 uM 25 ul 26 ul
Template DNA N/A 10 ul N/A 250 ul 260 ul
Nuclease Free
water N/A 13 ul N/A 325 ul 338 ul
Total 50 ul 1250 ul 1300 ul

Table 2: GoTaq2X Master Mix reaction