BIOL3900

Fall 2021

Omar Arias Gaguancela

 General Overview Part 1
1) RNA extraction
from plant tissues

2) Primer design and

bioinformatic tools

3) cDNA amplification

4) PCR amplification

5) Gel/PCR
purification

6) TA cloning and
Escherichia coli
transformation

7) Colony PCR
 2) Primer design and bioinformatic tools
https://blast.ncbi.nlm.nih.gov/Blast.cgi
2) Primer design and bioinformatic tools
 2) Primer design and bioinformatic tools
Rationale: Blastp to find Arabidopsis FAAH homologues in Soybean (Glycine max)
1) Paste AtFAAH sequence in “Enter Query Sequence”
2) Type Glycine max in “Organism”
3) Click on “BLAST”

1)

2)

3)
 2) Primer design and bioinformatic tools
Parameters to look
• Sorted based on E-values by default. The closer the e-value to cero the more likely the match is correct,
and the less the chances of selecting a false positive
• Percentage of identity: level of similarity between the input and output sequences
• Query Coverage: Percentage of input sequence that aligns to the output sequences
 2) Primer design and bioinformatic tools
1. Click on the top sequence
2. Click on sequence ID
3. Prompts to information page
4. Scroll down and look for CDS
(coding sequence), and click 1)
on it

2)
3)

4)
 2) Primer design and bioinformatic tools
1. Click on FASTA
2. It prompts to coding
sequence for GmFAAH1
3. Prompts to information
page
4. Look for ATG (start codon)
and TGA (stop codon). This
helps you to confirm you
are using a coding
sequence and not total
RNA

Start codon

Stop codon
 2) Primer design and bioinformatic tools
1. Select sequence
2. Copy and paste in a notepad document
3. Note that all sequences are labelled with a greater-than sing followed by the name of the gene (e.g.
“>GmFAAH1”)
4. Remove the STOP codon (TGA) of all your sequences – File name: FAAH sequences

STOP codon
 2) Primer design and bioinformatic tools
Forward primer:
1. Select a region of 18-30 nucleotides (e.g. 21 nucleotides) , and use the word count tool to confirm the number of
nucleotides
2. Copy and paste sequence in excel file titled GENWIZ_Oligo_Form_template, also, type a name for the primer in
the column “Oligo Name”
 2) Primer design and bioinformatic tools
Reverse primer:
1. Select a region of 18-30 nucleotides (e.g. 21 nucleotides) , and use the word count tool to confirm
the number of nucleotides
2. Copy and paste in Reverse Complement website
3. Copy and paste the outcome sequence in excel file
4. Name your reverse primer
5. Save excel file: Group number 00

1) 2) 3)

4)
 2) Primer design and bioinformatic tools

>GhFAAH1

ATGGGGAAAAAGCGTGTAATGACGCCAGCCAAGGACGTTGACTTATCTTCCATCAAATACGAACGTGAAATTGTG
TA-cloning
CAAGCTCCGCATTTGACTGGATTTGTATTTAGATTATTTGTGAGGATAATTGAAGCTCCGGTTATAGGCCCTATCAT
TATGAATATTCTGAAGAAAGAAAATAAAATGGATGAGATATTGCGGCACACTATGATACCTGAGGAACCCATGTTT
AAACCTGAATATCCACCTCAAGAAATGGAGCCTGGTGTTGTTGTCCTTGAGGAAGATGGGAGACCTGAAGATCGA
GTTGAGTATGCTCTGAAGTGTCTTCCACATTATGATGTTGCTGAATCATGGGAAAATTCATCTGCATCCTTTCGGTA
CTGGAAAATACGTGACTATGCTCATGCTTATCAATCTAGAAAAGTAACCCCATCTATGGTTGCCGAGCGCATTATCT
CAATTATAGAGGACAATGGAAGAAATAAACCTCCTACACCACTATTGATATCGTTTGATGCTGCAGGAGTACGGG
AGCAGGCGGCAGCATCTACTCAGAGGTTTGAAGCAGGAAATCCATTATCGATATTGGATGGAATTTTCATGGCCA
TCAAAGATGACATAGATTGTTATCCTCATCCATCTAAGGGTGCAACTACATGGATGCATGAGGTACGCACTGTAAA
GGAGGATGCAGTCTGTGTATCAAGACTGCGTACCTGTGGTGTCATATTCATCGGGAAGGCAAATATGCATGAGTT
AGGCATGGGTACAACAGGAAATAATCCTAATTTTGGAACTACAAGAAATCCTCATGCGCCTGACAGGTATACTGGT
GGATCTTCTTCAGGTCCAGCTGCAATTGTTGCTTCTGGACTATGTTCTGCTGCACTGGGAACTGATGGTGGAGGTT
CAGTCCGTATTCCTTCTTCCCTTTGTGGTGTGGTGGGATTTAAGACAACTTATGGGCGAACAAGCATGGAAGGGTC
ACTGTGTGATTCTGGGACCGTGGAAATTATTGGACCCATTGCTTCAACGGTGGAGGATGTTTTGCTGGTGTATTCT
GCAATGTTGGGTGCATCACCTGCAAATAGAATCAGTTTGAAACCGGCACCACCTTGTTTGCCAACTCTGTCATCCA
ATGATAATTCAAATGCCTTGGGATCTTTAAGAATTGGAAAGTACACCCCGTGGTTTAATGATGTACACTCAACTGA
AATCTCTGATAAATGTGAGGAAGCACTTAATCTGCTGTCTAAAGCACATGGTTGTGAAATGATAGAAATTGTTATA
CCAGAGCTTCTTGAGATGCGAACTGCCCATGTTGTTTCCATTGGCTCTGAATGCTTATGTTCACTGAATCCTGATTG
TGAAGACGGGAGAGGTGTAAATTTGACATATGATACTCGTACAAGTTTGGCACTTTTTCGGTCATTTACAGCAGCA
GATTATGTTGCAGCCCAATGTATCAGACGAAGGAATATGTATTACCACTTGGAGATTTTCAAGAAAGTGGATGTCA
TAGTAACGCCGACCACTGGCATGACAGCACCTATAATACCTCCCAGTGCTCTTAAAAGTGGTGAAACAGATATGCA
GACTACAGCTAACCTTATGCAGTTCGTTGTTCCTGCAAATCTTTTGGGATTCCCTTCCATTTCTGTCCCGGTTGGTTA
CGATAAAGAAGGACTTCCAATAGGCTTGCAAATAATGGGTCGACCATGGGCGGAAGCTACTATTCTGCGTGTAGC
CGCTGAAGTGGAGAAACTCTGTGGTGAGTCGAAGAAAAAACCCGTGTCATACTATGATGTTCTGAAGGCTAAA


Vector with
Sanger GhFAAH1
Sequencing sequence
to confirm Clones
that E. coli
with pTrcHis2-TOPO: GhFAAH1
Transformation
sequence has vector
no mutations
 2) Primer design and bioinformatic tools
1. Go to the following website: https://web.expasy.org/translate/
2. Copy/Paste target sequence
3. Click Translate – do not change the default settings
4. Copy and paste amino acid sequence labelled as Frame 1
5. Copy/Paste sequence in notepad and save file

1)

2)
 General Overview Part 1
1) RNA extraction
from plant tissues

2) Primer design and

bioinformatic tools

3) cDNA amplification

4) PCR amplification

5) Gel/PCR
purification

6) TA cloning and
Escherichia coli
transformation

7) Colony PCR
 3) cDNA amplification
• cDNA synthesis can be achieved with non or specific primers
 Non-specific primers: random hexamers (six random nucleotides that attach to
template) or oligo-dT primers (complementary to A tail)
 Specific primers: amplifies specific mRNAs from the total mRNA pool – more
restrictive

https://microbeonline.com/rt-pcr-principles-applications/
 3) cDNA amplification
1 RX- Volume n RX- Volume
Number Component (µL) (µL)
1 10X RT BUFFER 2  
2 25X dNTP Mix (100 mM) 0.8  
3 10X RT Random Primers 2  
4 MultiScribe Reverse Transcriptase 1  
5 Nuclease Free Water 4.2  
Final Volume 10  

1. Pipette each component into a 0.2 mL PCR tube (RT mix)

2. Pipette 10 µL of RNA sample into 0.2 mL PCR tube containing the RT mix
3. Seal the tubes and centrifuge (~30 seconds)
4. Place tubes in thermocycle for RT reaction
5. Reaction conditions:
Step1: 10 minutes at 25 °C
Step2: 120 minutes at 37 °C
Step 3: 5 minutes at 85 °C
Step 4: Short term storage at 4°C
Step 5: Long term storage at -20°C