Plasmids, primers (and beyond!)

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Plasmids, primers (and beyond!)
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Or how to make green mice.
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Foreign DNA can be incorporated into a cloning
vector (plasmid, phage, YAC or other) if both
foreign DNA and vector are cleaved with the same
restriction endonuclease. The pieces of DNA will
anneal, and then can be ligated using DNA
ligases. The desired fragment can be separated
from others using gel electrophoresis.
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Cleavage of DNA by restriction endonucleases,
followed by incorporation of foreign DNA into a
a plasmid using restriction endonucleases.
Insertion is facilitated by the annealing of
sticky ends of the restriction fragments.
(Some restriction enzymes give blunt ends
annealing then requires complementary blunts
ends. In either case, ligation is done by a DNA
ligase.
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Restriction enzymes Sticky ends vs. Nonsticky
ends
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Adding restriction sites

• We can design primers to add specific sequences
  of DNA to the beginning and end of our insert
  DNA.
• When we want to ligate a piece of DNA(in our case
  mefp-5) into a plasmid we use restriction
  enzymes. Restriction enzymes recognize and cleave
  certain palindromic pieces of DNA.

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Synthetic DNA with several cleavage sites can be
ligated into a vector to create a synthetic
polylinker site in the vector.
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Cohesive ends can be formed on a DNA fragment
(e.g., a synthetic oligonucleotide) by adding a
short segment using a DNA ligase (such as the one
from T4 ligase). The short segment contains a
cleavage site for a restriction enzyme, in this
case, EcoR1. EcoR1 forms an overhanging adhesive
site.
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(No Transcript)
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Tags?
Tags are added to your insert DNA in order to
purify the resultant protein after expression.
For instance, a his-tag, a sequence of 6
histidines, will cause the protein to stick to
certain kinds of chromatography columns. After
washing out all other proteins in your cell
lysate with buffer only your protein of interest
will remain. This protein can be removed from the
column with certain harsher chemicals.
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Adding tags

• Always make sure your insert is IN FRAME!
• For N-terminal tags, make sure to add a stop
  codon if there isnt one already.
• Alternatively, for C-terminal tags, make sure
  your insert doesnt have a stop codon.
• ON THE MAP
• From left to right the order of transcription is
  N-terminus to C-terminus

GAATTCGCGGCCGCTTCTAGGCATGAAGCTGTCGTGCATCGTCCTGGTCC
TCTTCCTGGTCACGCTGGCGGCTTACTCCGACGTGGGCTCGTCCTCGTCG
GAGGAGTATAAGGGCGGCTATTACCCAGGCAATGCCTATCACTATCATAG
TGGAGGCTCTTACCACGGCAGCGGCTATCACGGTGGCTACAAGGGCAAGT
ACTACGGTAAGGCGAAGAAATATTACTATAAGTACAAGAACTCGGGCAAG
TACAAATATCTCAAGAAGGCCCGCAAATACCATCGCAAGGGCTATAAGTA
CTACGGCGGCTCGTCGTACTAGTAGCGGCGGCTCCAGAGATCT
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Outline of a primer

• Forward primer
• Junk DNARestriction siteJunk DNA to keep in
  frame12 bp of sequence
• Reverse primer
• Take reverse complement of (Junk DNA12bp of
  sequenceJunk DNA to keep in framerestriction
  site)
• Why junk DNA at the beginning of your primer?
• Necessary in order for transcription machinery to
  bind to your sequence.

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pET 28a!
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A closer look

• Enzymes
• EcoRI GAAT TC
• NotIGCGGCCGC
• NcoI CCATGG
• NdeI CATATG

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N-terminal tag

• The beginning of mefp-5 is (in frame) GCA TGA AGC
  TGT CGT
• WE MUST PRESERVE THIS PATTERN.
• If we put an EcoRI site on the N-terminus of our
  mefp-5 insert DNA, our forward primer will be
• GAATTC GCATGAAGCTGTCGT
• as will the first
• When we cut mefp-5 the result is (in frame)
• AAT TCG CAT GAA GCT GTC GT
• ..WHICH IS OUT OF FRAME
• To fix this, add an extra piece of junk DNA to
  shift the reading frame back in its proper place
• GAATTC A GCATGAAGCTGTCGT
• ?AAT TCA GCATGAAGCTGTCGT
• Hooray! Back in our proper reading frame!

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N-terminal tagcontinued

• So what if we ONLY want an N-terminal tag, but NO
  C-terminal tag
• One word Stopcodons
• Insert a TAA at the N-terminal end of your
  sequence BEFORE the restriction site
• Plasmids like pET28a contain a stop codon AFTER
  the C-terminal histidine tag. This signals
  transcription to stop after the C-terminal
  histidine tag has been transcribed.
• We want transcription to stop BEFORE the C-term
  tag. So if we use NotI as our C-terminal
  restriction enzyme for our mefp-5 insert, the end
  of the sequence should look like (NotI is
  GCGGCCGC)
• TAC TAC GGC GGC TCG TCG TAA GCGGCCGC
• Which corresponds to the reverse primer
• GCGGCCGC TTA CGACGAGCCGCCGTAGTA
• of course its a real word.

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Adding a C-term tag

• His-tag must be in frame. But how?
• Use different enzymes
• Add extra bp

NotI Reverse TAC TAC GGC GGC TCG TCG
GCGGCCGC Primer GCGGCCGC CGACGAGCCGCCGTAGTA
EcoRI -Reverse TACTACGGCGGCTCGTCG T
GAATTC Primer GAATTC CGACGAGCCGCCGTAGTA
orangejunk DNA to keep in frame
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pGEX!
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• Straightforward N-terminal GST tag
• Keep in frame!
• Two variants we have, 4T-1 and 4T-3 just have
  different reading frames
• Stop codon is included at C-terminus
• Thrombin cleavage to cleave GST tag from your
  purified protein