Sequence and topology of TRPM8

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Sequence and topology of TRPM8
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TRPC3 TRPM2 TRPM8
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TRPM2 TRPM8
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chimera TRPM8
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1. Partial BamHI/XhoI digest
We dont want to cut here
BamHI
BamHI
2.7K
We want to cut here and here
1.5K
TRPM8
XhoI
pcDNA3
5.3K
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1. Partial Bam/Xho digest
3000 4000 5000 6000 8000 10000
Dilution of BamHI No BamHI 116 18 14 12
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2. Complete SpeI/XhoI digest
SpeI
2.9K
650 bp
SpeI
TRPM8
1.2K
SpeI
XhoI
pcDNA3
4.7K
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2. Complete Spe/Xho digest
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3. PCR amplification of S5-S6 linker from TRPM2 -
adding BamHI and SpeI sites
TRPM2 GTGGCCAAGCAGGCCATCCTC
V A K Q A I L
ATCCACAACGAGCGCCGGGTGGACTGGCTG I H N E R
R V D W L TTCCGAGGGGCCGTCTACCACTCCTACCTC
F R G A V Y H S Y L
ACCATCTTCGGGCAGATCCCGGGCTACATC T I F G Q
I P G Y I GACGGTGTGAACTTCAACCCGGAGCACTGC
D G V N F N P E H C
AGCCCCAATGGCACCGACCCCTACAAGCCT S P N G T
D P Y K P AAGTGCCCCGAGAGCGACGCGACGCAGCAG
K C P E S D A T Q Q
AGGCCGGCCTTCCCTGAGTGGCTGACGGTC R P A F P
E W L T V CTCCTACTCTGCCTCTACCTG L
L L C L Y L
5 primer AaaggATCCTCATCCACAACGAGCGCCGGGTG AAG
CAGGCCATCCTCATCCACAACGAGCGC K Q A I L I H
N E R
3 primer CCCTGAGTGGCTGACGGTCCTaCTAgTtTt TTCCCTG
AGTGGCTGACGGTCCTCCTACTCTGC F P E W L T V L
L L C 3 primer CCCTGAGTGGCTGACGGTCCcaCTAgTt
Tt 3'RC AAAACTAGTAGGACCGTCAGCCACTCAGGG
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4. After digestion of PCR product with
Bam/Spe, ligate with the Spe/Xho fragment - see
(2)
Some possible mis-ligations
(
)n
(
)n
How to prevent this? We dont allow Bam/Bam or
Xho/Xho ligations ligate in the presence of the
enzymes Bam and Xho
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4. Ligation of Bam/Spe and Spe/Xho fragments
We expect the following
200 bp
400 bp
1200 bp
1400 bp
2400 bp
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4. Ligation of Bam/Spe and Spe/Xho fragments
200 400 1200 1400 2400
200 1200
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5. Ligation of Xho/Bam and Bam/Xho fragments
BamHI
BamHI
1.5K (from step 4)
TRPM8
XhoI
XhoI
pcDNA3
8K (from step 1)
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Transform bugs (nonpathogenic strain of E. coli)
Plasmid DNA

• Add growth medium
• Incubate 1 hr to express antibiotic resistance

Heat shock 42 C 1 min
Spread on agar with antibiotic, leave overnight
Competent E. coli
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After transformation (next morning)
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Testing!!

• Cut the construct using the enzymes used to
  create it, check the bits are present
• Cut the insert out with enzymes other than those
  used to put it in. Check length.
• PCR amplify to check we have TRPM2 and not TRPM8
  sequence in the S5/S6 linker
• Sequence it directly

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Cut using enzymes that we used to make the
construct
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PCR amplification to check for M2 and not M8
sequence