Difference between revisions of "IPCR"
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(Inverted PCR/Biobrick Cloning) |
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1. Conduct regular PCR of insert/part B (eg. AdhE from J100107) using Prefix_For and Suffix_Rev primers | 1. Conduct regular PCR of insert/part B (eg. AdhE from J100107) using Prefix_For and Suffix_Rev primers | ||
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2. Purify PCR product and digest with XbaI + PstI | 2. Purify PCR product and digest with XbaI + PstI | ||
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3. Heat inactivate restriction enzymes at 80 C for 15 minutes | 3. Heat inactivate restriction enzymes at 80 C for 15 minutes | ||
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4. Conduct Inverted PCR of vector with part A (eg. promoter + C.dog in S05022) using Spe_Rev and Pst_For primers | 4. Conduct Inverted PCR of vector with part A (eg. promoter + C.dog in S05022) using Spe_Rev and Pst_For primers | ||
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5. Purify PCR product and digest with SpeI + PstI | 5. Purify PCR product and digest with SpeI + PstI | ||
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6. Heat inactivate restriction enzymes at 80 C for 15 minutes | 6. Heat inactivate restriction enzymes at 80 C for 15 minutes | ||
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7. Ligate the two PCR products and transform bacteria | 7. Ligate the two PCR products and transform bacteria | ||
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Prefix | Prefix | ||
Latest revision as of 19:49, 20 May 2013
INVERTED PCR/BIOBRICK CLONING
1. Conduct regular PCR of insert/part B (eg. AdhE from J100107) using Prefix_For and Suffix_Rev primers
2. Purify PCR product and digest with XbaI + PstI
3. Heat inactivate restriction enzymes at 80 C for 15 minutes
4. Conduct Inverted PCR of vector with part A (eg. promoter + C.dog in S05022) using Spe_Rev and Pst_For primers
5. Purify PCR product and digest with SpeI + PstI
6. Heat inactivate restriction enzymes at 80 C for 15 minutes
7. Ligate the two PCR products and transform bacteria
Prefix
GAATTCGCGGCCGCTTCTAGAG
CTTAAGCGCCGGCGAAGATCTC
Suffix TACTAGTAGCGGCCGCTGCAG ATGATCATCGCCGGCGACGTC
Prefix_For; TGCATGAATTCGCGG
Suffix_Rev; TGCATCTGCAGCGGC
Spe_Rev; GCATCCGCTACTAGTA
Pst_For; TGCATGCCGCTGCAG
