uence for one of these genes and asked to design a cloning strategy to generate a dsRNA expression construct to knock down that gene by RNAi . 1. What two restriction sites are you going to use to clone your PCR product into the pL4440 plasmid? What are their DNA sequence? 2. How would you go about cloning this amplified DNA into pL4440? Using your knowledge of cloning list 5 important aspects of the method.

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You were provided with the genetic sequence for one of these genes and asked to design a cloning strategy to generate a dsRNA expression construct to knock down that gene by RNAi . 1. What two restriction sites are you going to use to clone your PCR product into the pL4440 plasmid? What are their DNA sequence? 2. How would you go about cloning this amplified DNA into pL4440? Using your knowledge of cloning list 5 important aspects of the method.
L
How to design the RNAi expression construct
It is possible to knock down any gene in the C. elegans genome using this RNAi feeding method.
Before you start, you need to design and make your RNA expression construct. You will use
the F27C1.8 gene as an example and design a cloning strategy to do this.
"A region of the gene of interest is cloned into the multiple cloning site (MCS) region of plasmid L4440. Treatment
with IPTG induces expression from the T7 promoter and produces complementary RNAS, which join up to become
dsRNAS."
e
This is the sequence of F27C1.8 taken from Wormbase (so you can see what it looks like).<
Unspliced + UTR (855bp)
> Unspliced UTR (855bp)
1 ATGGTAAAGG CCOTCGTCGG ATTCGGCGCT GCATGCGGAA TCTCTGCTAT
51 CGTTGCTTGC CTTTGGGCTG CACTTGTCAT CACAAATGAC ATCAATGACA
101 TGTATGATGA TOTGATGGGA GAGCTCGGAG GATTCAGAGA TATCTCTGAT
151 GACACTTGGG GAACCCTTCT CGACGTTCGT CACGGAGCCG GAGAGTCTGC
201 TGAGCAATAC GTTCGTGGAA TCTTCOGACO TCACAAGCGT TCCAACAGCC
251 AATOCTCTTG COGACTTCCA TCTCAAGGAT GCCCAGCCGG AGCTCCAGGA
301 AACCCAGGAG CCCCAGGAGA GCCAGGAGGC ACTGGACCAG ACGGAAAGAA
351 COGACCAACT GGACTTCCAG GACTTAACAT TCCAATTCCA AATGACTTCC
401 CTAAGGAGTO CATCAAGTGC CCAGCTGGAC CACCAGGACA AGATGGACTT
451 CCAGGACAAG AAGGATTCCA AGGACTTCCA 60AGACGCTG GAAAGCGTGG
501 AACCCCAGGA AAGGACGGAG AGCCAGGACG TOTTGGAGAT ATTGGAGATC
551 AAGGAACTCC AGGACAAGAC GGACAACCAG GACTTGCTGG ACCACCAGGA
601 COCGATOGAC TTACCGGAAA GGGACAACCA 6GAGTCGCTG GACGCCCAGG
651 AATOCCAGGA CCACGTGGAG AGCCAGGAAA CAACGGAAAT CCAGGAGAGG
701 AAGGACAAAC TGGAGCCCAG G6ACCAACTO GACAGCCAGG AAAGGACGGA
751 TTCAACOGAA ACGACGGAAC TCCAGGACAA GCTGGACCAC AAGGAGCCGT
801 TGGAGCCGAT GCCGAATACT GCCCATGCCC AGAGAGAAAG CGCAGACGCO
851 TCTAA
Legends
A Exon
H
From the above sequence I have copied the first 500bp of the sequence into Word.
ATGGTAAAGGCCGTCGTCGGATTCGGCGCTGCATGCGGAATCTCTGCTATCGTTGCTTGCCTTTGGGCTGCACTTGTCATCACAAA
TGACATCAATGACATGTATGATGATGTGATGGGAGAGCTCGGAGGATTCAGAGATATCTCTGATGACACTTGGGGAACCCTTCTCG
ACGTTCGTCACGGAGCCGGAGAGTCTGCTGAGCAATACGTTCGTGGAATCTTCGGACGTCACAAGCGTTCCAACAGCCAATGCTCT
TGCGGACTTCCATCTCAAGGATGCCCAGCCGGAGCTCCAGGAAACCCAGGAGCCCCAGGAGAGCCAGGAGGCACTGGACCAGACGG
AAAGAACGGACCAACTGGACTTCCAGGACTTAACATTCCAATTCCAAATGACTTCCCTAAGGAGTGCATCAAGTGCCCAGCTGGAC
CACCAGGACAAGATGGACTTCCAGGACAAGAAGGATTCCAAGGACTTCCAGGAGACGCTGGAAAGCGTGG
(
1) You now need to design primers to amplify this 500bp region of the DNA. Write out
the primer sequences in the correct orientation.<
(
2) Decide on two restriction sites that you can use to clone this into pL4440's MCS.
Identify their sequence.<
Tip: The plasmid map is in Figure 3, details of restriction site sequences can be found
at https://enzymefinder.neb.com/#!#nebheader
(
3) Add the restriction site DNA sequences to the correct end of each primer.<
E
Transcribed Image Text:L How to design the RNAi expression construct It is possible to knock down any gene in the C. elegans genome using this RNAi feeding method. Before you start, you need to design and make your RNA expression construct. You will use the F27C1.8 gene as an example and design a cloning strategy to do this. "A region of the gene of interest is cloned into the multiple cloning site (MCS) region of plasmid L4440. Treatment with IPTG induces expression from the T7 promoter and produces complementary RNAS, which join up to become dsRNAS." e This is the sequence of F27C1.8 taken from Wormbase (so you can see what it looks like).< Unspliced + UTR (855bp) > Unspliced UTR (855bp) 1 ATGGTAAAGG CCOTCGTCGG ATTCGGCGCT GCATGCGGAA TCTCTGCTAT 51 CGTTGCTTGC CTTTGGGCTG CACTTGTCAT CACAAATGAC ATCAATGACA 101 TGTATGATGA TOTGATGGGA GAGCTCGGAG GATTCAGAGA TATCTCTGAT 151 GACACTTGGG GAACCCTTCT CGACGTTCGT CACGGAGCCG GAGAGTCTGC 201 TGAGCAATAC GTTCGTGGAA TCTTCOGACO TCACAAGCGT TCCAACAGCC 251 AATOCTCTTG COGACTTCCA TCTCAAGGAT GCCCAGCCGG AGCTCCAGGA 301 AACCCAGGAG CCCCAGGAGA GCCAGGAGGC ACTGGACCAG ACGGAAAGAA 351 COGACCAACT GGACTTCCAG GACTTAACAT TCCAATTCCA AATGACTTCC 401 CTAAGGAGTO CATCAAGTGC CCAGCTGGAC CACCAGGACA AGATGGACTT 451 CCAGGACAAG AAGGATTCCA AGGACTTCCA 60AGACGCTG GAAAGCGTGG 501 AACCCCAGGA AAGGACGGAG AGCCAGGACG TOTTGGAGAT ATTGGAGATC 551 AAGGAACTCC AGGACAAGAC GGACAACCAG GACTTGCTGG ACCACCAGGA 601 COCGATOGAC TTACCGGAAA GGGACAACCA 6GAGTCGCTG GACGCCCAGG 651 AATOCCAGGA CCACGTGGAG AGCCAGGAAA CAACGGAAAT CCAGGAGAGG 701 AAGGACAAAC TGGAGCCCAG G6ACCAACTO GACAGCCAGG AAAGGACGGA 751 TTCAACOGAA ACGACGGAAC TCCAGGACAA GCTGGACCAC AAGGAGCCGT 801 TGGAGCCGAT GCCGAATACT GCCCATGCCC AGAGAGAAAG CGCAGACGCO 851 TCTAA Legends A Exon H From the above sequence I have copied the first 500bp of the sequence into Word. ATGGTAAAGGCCGTCGTCGGATTCGGCGCTGCATGCGGAATCTCTGCTATCGTTGCTTGCCTTTGGGCTGCACTTGTCATCACAAA TGACATCAATGACATGTATGATGATGTGATGGGAGAGCTCGGAGGATTCAGAGATATCTCTGATGACACTTGGGGAACCCTTCTCG ACGTTCGTCACGGAGCCGGAGAGTCTGCTGAGCAATACGTTCGTGGAATCTTCGGACGTCACAAGCGTTCCAACAGCCAATGCTCT TGCGGACTTCCATCTCAAGGATGCCCAGCCGGAGCTCCAGGAAACCCAGGAGCCCCAGGAGAGCCAGGAGGCACTGGACCAGACGG AAAGAACGGACCAACTGGACTTCCAGGACTTAACATTCCAATTCCAAATGACTTCCCTAAGGAGTGCATCAAGTGCCCAGCTGGAC CACCAGGACAAGATGGACTTCCAGGACAAGAAGGATTCCAAGGACTTCCAGGAGACGCTGGAAAGCGTGG ( 1) You now need to design primers to amplify this 500bp region of the DNA. Write out the primer sequences in the correct orientation.< ( 2) Decide on two restriction sites that you can use to clone this into pL4440's MCS. Identify their sequence.< Tip: The plasmid map is in Figure 3, details of restriction site sequences can be found at https://enzymefinder.neb.com/#!#nebheader ( 3) Add the restriction site DNA sequences to the correct end of each primer.< E
L
4) List the steps required to clone the PCR prduct into the pL4440 plasmid. <
Exact recipes and details of PCR mixes + machine conditions etc are not required, just
try and think about the whole procedure from start to finish.
E
(
Xbal
Spel
17
promoter
L4440
-2.790 bp
Amp
Eagl
T7 promoter coaGCACATCTOATATCATCGATGAATTCGAGCTCCACCOCOGTOGCGGCCGCTC
Not
Neo Nhe!
MCS 77
promoter
TAGATCTAGAAGPAGTOGATCCACCOUNCATOGCTAGOCACGTGACGCOTOdcccccdGGCTGCA
Sal I
Mly 1
Xmal
Sma
GGAATTOGATATCAATTATCGATACCO FEGACCICAGOO
Hine
Hind
Xho
Ард Ken 1
CCOOTACCCAATT T7 promoter
Figure 3: plasmid L4440 with details of it's multiple cloning site (MCS). You will need to clone, using restriction
sites, your DNA sequence into the MCS region of the plasmid. This diagram shows you the restriction sites you
can use.
专
Transcribed Image Text:L 4) List the steps required to clone the PCR prduct into the pL4440 plasmid. < Exact recipes and details of PCR mixes + machine conditions etc are not required, just try and think about the whole procedure from start to finish. E ( Xbal Spel 17 promoter L4440 -2.790 bp Amp Eagl T7 promoter coaGCACATCTOATATCATCGATGAATTCGAGCTCCACCOCOGTOGCGGCCGCTC Not Neo Nhe! MCS 77 promoter TAGATCTAGAAGPAGTOGATCCACCOUNCATOGCTAGOCACGTGACGCOTOdcccccdGGCTGCA Sal I Mly 1 Xmal Sma GGAATTOGATATCAATTATCGATACCO FEGACCICAGOO Hine Hind Xho Ард Ken 1 CCOOTACCCAATT T7 promoter Figure 3: plasmid L4440 with details of it's multiple cloning site (MCS). You will need to clone, using restriction sites, your DNA sequence into the MCS region of the plasmid. This diagram shows you the restriction sites you can use. 专
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