BME

MOLECULAR BIOLOGYEXPERIMENTPCR & SEEDINGSKKU BME

3RD GRADE, 2ND SEMESTER

FOR FUN

PCR & SEEDING

• PCR: polymerase chain reaction

• Electrophoresis

• Seeding

• These are for amplifying DNA and cell…

PCR – WHAT DO YOU NEED?

• Template

• Primers

• Polymerase

• dNTP

• Mg2+ --- What’s the function of this?

• Buffer

PCR – WHAT SHOULD YOU KNOW?

• Temperature

• Step-by-step temperature!

1. Denaturation2. Annealing3. Polymerization

PCR CYCLE

PHASE OF PCR IDEA!How can we use the linear phase!!!

MAKING PRIMERS

• What should you consider for making the primers?

1. Sequence

3. Annealing temperature

2. Length

FIND THE DNA SEQUENCE!

• https://www.addgene.org/vector-database/2487/

EGFP DNA SEQUENCE

5’-ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA ACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAG TGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACG TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGG TGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACAC CCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAA GACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCA TGGACGAGCTGTACAAGTAA-3’ What can you see in the sequence???

1. Start codon2. Stop codon3. Correct frame for the Translation?

https://www.ncbi.nlm.nih.gov/nuccore/U55762.1

PCR PRIMERS

Template3’5’

EGFP DNA SEQUENCE

5’-ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA ACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAG TGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACG TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGG TGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACAC CCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAA GACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCA TGGACGAGCTGTACAAGTAA-3’ What can you see in the sequence???

1. Start codon2. Stop codon3. Correct frame for the Translation?

https://www.ncbi.nlm.nih.gov/nuccore/U55762.1

ANNEALING TEMPERATURE

POLYMERASES

• Taq polymerase

• Pfu

Taq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Chien et al. in 1976. Its name is often abbreviated to Taq Pol or simply Taq. It is frequently used in the polymerase chain reaction (PCR), a method for greatly amplifying the quantity of short segments of DNA.

MAKING PRIMERS

• What should you consider for making the primers?

1. Sequence

3. Annealing temperature

2. Length

Question> How to ligase the insert to vector?Hint> Endonuclease reaction and sticky end

ENDONUCLEASE AND STICKY ENDS

PCR product

EGFP DNA SEQUENCE

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA ACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAG TGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACG TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGG TGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACAC CCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAA GACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCA TGGACGAGCTGTACAAGTAA What can you see in the sequence???

1. Start codon2. Stop codon3. Correct frame for the Translation?

https://www.ncbi.nlm.nih.gov/nuccore/U55762.1

VECTOR – WHICH ENZYME SITES?

What is your final purpose?=>Protein expression!

What should you consider?

PRIMER CONSIDERATION – WHICH ENZYME SITES?

What is the MCB?

What should you consider for using the restriction enzyme sites?

WHICH ENZYME DO YOU CHOOSE?

• Making your own primers for GST-EGFP expression

• What should you consider? – summary

• Template sequence and annealing temperature

• Start and stop codons

• Restriction enzyme

• Extra sequence for enzyme reaction

aTemplate

5’ 3’

Is it working?

DNA ELECTROPHORESIS

MAKING AGAROSE GEL

• 1% agarose gel in TAE buffer

• Making 1XTAE buffer (500 ml)

• 25 ml 1xTAE buffer + ____ g agarose?

• Microwave for 3 min

• Add SYBR Safe dye – intercalating cheminal

BE CAREFUL…

• SYBR Green I is marketed as a replacement for the potential human mutagen ethidium bromide, as both safer to work with and free from the complex waste disposal issues of ethidium. However any small molecule capable of binding DNA with high affinity is a possible carcinogen, including SYBR Green.

SEEDING

E.COLI SEEDING FOR DNA PREP.

• E. coli growth curve (You already saw the growth pattern of the E.coli on plate!)

• Anaerobic and aerobic respirations – amount of air?

• Protection from contamination - antibiotics

E. COLI GROWTH CURVE

• Please how the E.coli grows in the plate and LB broth.

SEEDING BY LOOP

• Prepare 5 ml LB broth + antibiotics (amp for pGEX vector)

• Choose one colony and mark by a pen

• Sanitization by flare

• Scoop one colony

• Seed in the LB broth

HOMEWORK

• Please find what is the pfu polymerase and difference from Taq polymerase

• What is the function of Mg2+ in PCR reaction?

• What are the blunt and sticky ends of DNA?

• Please find the differences of anaerobic and aerobic respirations of E. coli.

• What’s the coil and supercoil structures of plasmid?

• What is the Quantitative PCR, or real-time PCR? (hint, linear phase of PCR)

• Please find intercalating chemicals, especially EtBr and CYBR green.

• Please go to the web site, https://www.addgene.org/vector-database/2487/ and find and paste the CMV promoter sequence.

NEXT WEEK

• Mini prep

• Endonuclease reaction