Restriction and PCR Analysis - Online

The goals of this exercise are to:
  1. Determine whether the restriction patterns that you observed are expected based on the DNA sequences.
  2. Design a PCR experiment to verify the size and orientation of the inserted DNA fragments
  3. The Sequence extractor program will be used to map primer sequences onto the expected DNA sequences and generate virtual PCR fragments.
  4. The Primer3 program will be used to pick new primers.
  5. Ultimately we want 2 primers that flank the location of the insert (these will allow us to determine the size of the insert)and one that will hybridize to the insert to generate a PCR fragment (along with one or both of the flanking primers) that will uniquely identify the insert orientation.
  6. Email me the a note containing :
    1. The number(s) of the clones that contain inserts and whether the fragments are as predicted by NEBcutter.
    2. The primers that will be used to verify the clones and the expected PCR fragment sizes
Use each of the plasmid sequences in turn as inputs for the NEB cutter program to verify the expected restriction fragment lengths.
Clones 1-40 have Cro DNA inserted (Sequences 2 or 3 below).
Clones 40-120 have scCro DNA inserted (Sequences 4 or 5 below).

NEBcutter

  1. Open the fasta format files in a new window and copy the entire text
  2. Open the NebCutter site in a new window and paste the DNA sequence into the box
  3. Select the circular option and hit submit
  4. After some time a graphical restriction map will appear, select custom digest from the options at the bottom
  5. Either scroll through the list to select PstI and EcoRI or type the enzyme names in the "Pick this enzyme:" box
  6. Hit the digest button, a circular map appears with only
  7. Hit the view gel button to see a virtual gel
  8. You can change the agarose percentage and add DNA markers for size comparison.

Do the fragment sizes that you saw on the gel match up with those given by NEB cutter? If not, the clones that we have obtained may not be quite what we anticipated.

Another way to analyze DNA is by PCR. The Sequence Extractor program will give a text-based map of the DNA sequences and allow one to map the positions of PCR primers onto the sequence. WARNING - Formatting problems in ie7 use another browser (Firefox works).

Sequence Extractor

  1. Open the fasta format files in a new window and copy the entire text
  2. Open the Sequence Extractor site in a new window and paste the DNA sequence into the upper box
  3. Open the Primers file in a new window and paste the primer sequences into the lower box
  4. Select the circular option and hit submit
  5. After some time a text restriction map will appear
  6. Clicking on two restriction sites or primers will "extract" the intervening sequence into a new pop up window
  7. Try to find a one pair of primers that flank the insert to determine the size of the insert
  8. Try to find a second pair of primers to determine the orientation of the insert
  9. If appropriate primers are not in the list pick new primers with Primer3 (below).

Primer3

  1. Open the fasta format files in a new window and copy the entire text
  2. Open the Primer3 site in a new window and paste the DNA sequence into the upper box
  3. Hit submit and the program will pick PCR primers based on the default parameters
  4. Follow the instructions to identify desired targets and PCR product sizes

Plasmid Sequences

  1. Cro substitution mutant - tryptophan for phenylalanine at 58 Reverse Complement pUCroF58W_RSrc.fasta pUCroF58W_RSrc.fasta.
  2. pAH125 (PDF summary pAH125.pdf | genbank sequencepAH125.genbank | ncbi link) is a promoterless lacZ reporter plasmid, with kanR, OriR and Att lambda for generation of single copy integrants
  3. pAH125 Pst-Sal-lacI(rc)-Sph-OPO-Xba...-EcoRI-BamHI-lacZ pAH125laciOPO.fasta. lacI (rev. comp.) + lambda PR/lac O promoter inserted between the PstI and XbaI sites of pAH125
  4. As above plus "single-chain" Cro gene. pAH125 Pst-Sal-lacI(rc)-Sph-OPOXba-scCro-XhoI-EcoRI-BamHI-lacZ pAH125lacI_OPO_scCro.fasta.

Online DNA sequence analysis resources