SBB13Ntbk-Ted Chavkin: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
No edit summary |
||
Line 1: | Line 1: | ||
__NOTOC__ | __NOTOC__ | ||
==13:52, 14 March 2013 (EDT)== | |||
Running Zymo Cleanup on quiC2 PCA2<br> | |||
<br> | |||
Running EcoRI/BamHI digestion on quiC2 PCA2:<br> | |||
<pre> | |||
8uL of eluted PCR product | |||
1uL of NEB Buffer 2 | |||
0.5uL EcoRI | |||
0.5uL BamHI | |||
<math>37^0</math>C for 1 hour | |||
</pre> | |||
==[[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:<br> | Running Zymo Cleanup on quiC PCA1:<br> | ||
<pre> | <pre> | ||
Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction. | Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction. | ||
Transfer into the Zymo column (small clear guys) | Transfer into the Zymo column (small clear guys) | ||
Line 18: | Line 28: | ||
</pre> | </pre> | ||
Running PCA2:<br> | Running quiC PCA2:<br> | ||
<pre> | <pre> | ||
Recipe | Recipe | ||
Line 36: | Line 46: | ||
==[[User:Theodore A Chavkin|Theodore A Chavkin]] 11:51, 5 March 2013 (PST)== | ==[[User:Theodore A Chavkin|Theodore A Chavkin]] 11:51, 5 March 2013 (PST)== | ||
Running PCA1:<br> | Running quiC PCA1:<br> | ||
<pre> | <pre> | ||
Line 68: | Line 78: | ||
AGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCATGAGATCTGCATCGTCTCAATACGGAACAAACGTTAGAACTATGACCTTGCCAGGTTTAGTCTTGCAAGATGTTAATGGTGTTACTTTGAAGGGATTAGACGTTCATAGGTTCTGTATAGGAGTCTTAGTTAACAGATCATCTAACAATTTAATTCAACACAATAGAATATCAAACAATTACGGAGGTGCCGGTGTCATGATTACTGGTGATGATGGTAAAGGTAACCCAACTTCTACCACAACTAACAACAATAAAGTCTTAGATAATGTCTTTATTGATAATGGTGATGGTTTAGAGTTAACTAGAGGTGCTGCTTTTAATTTAATTGCTAATAATTTATTCACATCAACTAAGGCTAATCCTGAACCATCACAAGGTATCGAAATTTTGTGGGGTAACGATAATGTGAGACGGCATGGATCCTAACTCGAGCTGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAAT | AGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCATGAGATCTGCATCGTCTCAATACGGAACAAACGTTAGAACTATGACCTTGCCAGGTTTAGTCTTGCAAGATGTTAATGGTGTTACTTTGAAGGGATTAGACGTTCATAGGTTCTGTATAGGAGTCTTAGTTAACAGATCATCTAACAATTTAATTCAACACAATAGAATATCAAACAATTACGGAGGTGCCGGTGTCATGATTACTGGTGATGATGGTAAAGGTAACCCAACTTCTACCACAACTAACAACAATAAAGTCTTAGATAATGTCTTTATTGATAATGGTGATGGTTTAGAGTTAACTAGAGGTGCTGCTTTTAATTTAATTGCTAATAATTTATTCACATCAACTAAGGCTAATCCTGAACCATCACAAGGTATCGAAATTTTGTGGGGTAACGATAATGTGAGACGGCATGGATCCTAACTCGAGCTGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAAT | ||
</pre> | </pre> | ||
Revision as of 10:52, 14 March 2013
13:52, 14 March 2013 (EDT)
Running Zymo Cleanup on quiC2 PCA2
Running EcoRI/BamHI digestion on quiC2 PCA2:
8uL of eluted PCR product 1uL of NEB Buffer 2 0.5uL EcoRI 0.5uL BamHI <math>37^0</math>C for 1 hour
Theodore A Chavkin 10:08, 12 March 2013 (PST)
Running Zymo Cleanup on quiC PCA1:
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
Running quiC PCA2:
Recipe 1. 1 ul each outer oligo (10 uM) 2. 1 ul purified pca product 3. .5 ul phusion 4. 10 ul 5x phusion buffer 5. 5 ul 2mM dNTPs 6. 32.5 ul H2O Program 1. 2 min initial denature at 94oC 2. 30 sec denature at 94oC 3. 30 sec anneal at 60oC [This should be high, as your outer oligos now have a huge overlap with the correct product] 4. 30 sec extension at 68oC 5. repeat 2-4 30 times total
Theodore A Chavkin 11:51, 5 March 2013 (PST)
Running quiC PCA1:
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
Construction File for pTC001:
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