SBB13Ntbk-Ted Chavkin: Difference between revisions
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[[User:Theodore A Chavkin|Theodore A Chavkin]] 10:08, 12 March 2013 (PST) | [[User:Theodore A Chavkin|Theodore A Chavkin]] 10:08, 12 March 2013 (PST) | ||
Running Zymo Cleanup on PCA1 | <> | ||
Running Zymo Cleanup on PCA1 | |||
<pre> | <pre> | ||
Regular Zymo Cleanup | Regular Zymo Cleanup | ||
Revision as of 10:17, 12 March 2013
Theodore A Chavkin 10:08, 12 March 2013 (PST) <> Running Zymo Cleanup on PCA1
Regular Zymo Cleanup The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction. It also will remove the buffer and restriction enzymes from a restriction digest reaction. Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction. Transfer into the Zymo column (small clear guys) spin through, discard waste. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol) spin through, discard waste. Add 200 uL of Zymo Wash Buffer spin through, discard waste. spin for 90 seconds, full speed to dry. elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction
Theodore A Chavkin 11:51, 5 March 2013 (PST)
PCA1 quiC-2_oligo[1-16] (471bp, pca1) PCA2 quiC-2_oligo3/6 on pca1 (471bp, quiC2) Digest quiC2 (EcoRI/BamHI, 426+26+19bp, L, quiC2dig) Digest pBca9145-Bca114#5 (EcoRI/BamHI, 2057+910bp, L, vectdig) Ligate quiC2dig + vectdig (pTC001) >quiC2 ATATAGATGCCGTCCTAGCGAATTCATGAGATCTGCATCGTCTCAATACGGAACAAACGTTAGAACTATGACCTTGCCAGGTTTAGTCTTGCAAGATGTTAATGGTGTTACTTTGAAGGGATTAGACGTTCATAGGTTCTGTATAGGAGTCTTAGTTAACAGATCATCTAACAATTTAATTCAACACAATAGAATATCAAACAATTACGGAGGTGCCGGTGTCATGATTACTGGTGATGATGGTAAAGGTAACCCAACTTCTACCACAACTAACAACAATAAAGTCTTAGATAATGTCTTTATTGATAATGGTGATGGTTTAGAGTTAACTAGAGGTGCTGCTTTTAATTTAATTGCTAATAATTTATTCACATCAACTAAGGCTAATCCTGAACCATCACAAGGTATCGAAATTTTGTGGGGTAACGATAATGTGAGACGGCATGGATCCTGGGCACAGGAAAGATACTT >pTC001 AGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCATGAGATCTGCATCGTCTCAATACGGAACAAACGTTAGAACTATGACCTTGCCAGGTTTAGTCTTGCAAGATGTTAATGGTGTTACTTTGAAGGGATTAGACGTTCATAGGTTCTGTATAGGAGTCTTAGTTAACAGATCATCTAACAATTTAATTCAACACAATAGAATATCAAACAATTACGGAGGTGCCGGTGTCATGATTACTGGTGATGATGGTAAAGGTAACCCAACTTCTACCACAACTAACAACAATAAAGTCTTAGATAATGTCTTTATTGATAATGGTGATGGTTTAGAGTTAACTAGAGGTGCTGCTTTTAATTTAATTGCTAATAATTTATTCACATCAACTAAGGCTAATCCTGAACCATCACAAGGTATCGAAATTTTGTGGGGTAACGATAATGTGAGACGGCATGGATCCTAACTCGAGCTGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAAT Protocol: 1. 38 uL ddH2O 2. 5 ul 10x expand buffer 3. 5 ul 2mM dNTPs 4. 1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks) 5. 0.75 ul Expand polymerase Program: 1. 2 min initial denature at 94oC 2. 30 sec denature at 94oC 3. 30 sec anneal at 55oC [This should be the overlap temp of your oligos - vary as needed] 4. 30 sec extension at 72oC 5. repeat 2-4 30 times total
A wild conrad appears!! File:Conradmckinnon 1334093179 49.jpg
