JCAOligoTutorial2b prompt

From OpenWetWare
Jump to navigationJump to search
I am trying to understand a tutorial about the recombinant DNA technique SOEing.  The technique is used to silently remove a restriction site when cloning a gene to make a BioBrick part.  The experiment involves design of 2 oligos to PCR the region up to the restriction site, and then 2 oligos to amplify the region after the site.  Those two PCRs are joined in a SOEing reaction to make a new PCR product without the restriction site.  This is then then digested with restriction enzymes at sites incorporated into the ends of the oligos. The material is then ligated into a similarly-digested plasmid vector to make the final product. The entire experiment is expressed in this format:
"PCR    ca1110F  ca1111R     pBACr899  A
PCR    ca1111F  ca899R      pBACr899  B
SOE    ca1110F  ca899R      A   B     pcrpdt
Digest pcrpdt   EcoRI,BamHI 1         pcrdig
Digest pBca1100 EcoRI,BamHI 1         plasdig
Ligate pcrdig   plasdig               pBca1100-Bca1111

# Sequences
oligo ca1110F ctctggaattcatgAGATCTGCGATCCCGCGAAGAACC
oligo ca1111F catgaagtagatTtcgccaatgtc
oligo ca1111R gacattggcgaAatctacttcatg
oligo ca899R  GCAAAggatccTCTATGGTACTCGTGATGGC"

Following this spec:
# CF Shorthand Specification

## Layer 0: Basic Concept

A Construction File (CF) is a list of Steps, with each Step representing a specific operation in a molecular biology experiment. Each Step is written on a new line. Parameters are separate by whitespace, preferably TSV. The Step includes the names of input DNA sequence(s), non-sequence parameters, and ends with the name of the product DNA sequence. The input sequences can refer to products of previous steps. The product is the output of the operation.

In addition, a CF can include sequences in the form of 'name sequence' lines.

Comment lines follow '#', '//', or '/*comment*/' syntax.

## Layer 1: Core Operations

This layer defines a core set of operations: PCR, Digest, Ligate, GoldenGate, Gibson, and Transform. Each operation has specific parameters and (optionally) a product name.

## Examples

- `PCR ForwardPrimer ReversePrimer Template ProductName`
    - ForwardPrimer, ReversePrimer, Template: Names of DNA sequences
- `Digest DNA Enzymes FragmentSelection  ProductName`
    - DNA: Name of the DNA sequence to be digested
    - Enzymes: List of enzymes to be used, e.g. EcoRI,BamHI
    - FragmentSelection: Index indicating the chosen DNA fragment following digestion, numbered from zero beginning from the first enzyme's initial cut site, usually 1.
- `Ligate Fragment1 Fragment2 ProductName`
    - Fragment1, Fragment2: Names of DNA sequences
- `GoldenGate Fragment1 Fragment2 Enzyme ProductName`
    - Fragment1, Fragment2: Names of DNA sequences
    - Enzyme: Type IIS-like enzyme to be used along with ligase. e.g. BsaI
- `Gibson Fragment1 Fragment2 ProductName`
    - Fragment1, Fragment2: Names of DNA sequences
- `Transform Plasmid Host Antibiotic Temperature ProductName`
    - Plasmid: Name of the DNA sequence
    - Host: Bacterial strain
    - Antibiotics: Antibiotics used, e.g. Amp,Kan
    - Temperature: Incubation temperature in Celsius, e.g. 37

I need to predict the sequence of each PCR product and the plasmid product. I also need to design another experiment with a different template to remove a different restriction site using SOEing.  Can you help me understand this?