SBB13Ntbk-Robert Chen: Difference between revisions
Robert Chen (talk | contribs) (New page: '''2013 03 06:''' Designed oligos for PCA1 on CCOMT-1 CCOMT1_Synthon PCA for oligos 1-11, 13-14, 16 (407bp, PCA1_pdt) PCA for oligos 12,15, PCA1_pdt (462bp, PCA2_pdt) Digest P...) |
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'''2013 03 06:''' | '''2013 03 06:''' | ||
Designed oligos for PCA1 on CCOMT-1 | Designed oligos for PCA1 on CCOMT-1 | ||
CCOMT1_Synthon | <pre>CCOMT1_Synthon | ||
PCA for oligos 1-11, 13-14, 16 (407bp, PCA1_pdt) | PCA for oligos 1-11, 13-14, 16 (407bp, PCA1_pdt) | ||
PCA for oligos 12,15, PCA1_pdt (462bp, PCA2_pdt) | PCA for oligos 12,15, PCA1_pdt (462bp, PCA2_pdt) | ||
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CCOMT-1_oligo_8: ATTCTTAACTAAATATTTTGCTACTGTTGCTAATCCATACAATCTTTGTACCTT | CCOMT-1_oligo_8: ATTCTTAACTAAATATTTTGCTACTGTTGCTAATCCATACAATCTTTGTACCTT | ||
CCOMT-1_oligo_16: AACAGTAGCAAAATATTTAGTTAAGAATGAAGATGGTGTTTCTATGAGACGGCA | CCOMT-1_oligo_16: AACAGTAGCAAAATATTTAGTTAAGAATGAAGATGGTGTTTCTATGAGACGGCA | ||
CCOMT-1_oligo_15: AAGTATCTTTCCTGTGCCCAGGATCCATGCCGTCTCATAGAAACACCATCTTC | CCOMT-1_oligo_15: AAGTATCTTTCCTGTGCCCAGGATCCATGCCGTCTCATAGAAACACCATCTTC</pre> | ||
PCA1_pdt: AGATCTGCATCGTCTCATCGGTCTCCTATGGGATCTACAGGTGAGACACAAATAACTCCTACACATATTTCTGATGAAGAAGCTAACTTATTTGCAATGCAGTTGGCCTCTGCTTCAGTTTTGCCTATGATTTTGAAATCAGCTTTAGAATTGGACTTATTAGAGATTATTGCTAAGGCAGGTCCTGGAGCACAAATTTCACCTATCGAAATTGCCTCACAATTACCAACAACAAACCCTGATGCCCCAGTAATGTTGGATAGGATGTTAAGGTTATTAGCTTGCTATATTATATTGACATGCTCTGTAAGAACACAACAAGACGGTAAGGTACAAAGATTGTATGGATTAGCAACAGTAGCAAAATATTTAGTTAAGAATGAAGATGGTGTTTCTATGAGACGGCA | PCA1_pdt: AGATCTGCATCGTCTCATCGGTCTCCTATGGGATCTACAGGTGAGACACAAATAACTCCTACACATATTTCTGATGAAGAAGCTAACTTATTTGCAATGCAGTTGGCCTCTGCTTCAGTTTTGCCTATGATTTTGAAATCAGCTTTAGAATTGGACTTATTAGAGATTATTGCTAAGGCAGGTCCTGGAGCACAAATTTCACCTATCGAAATTGCCTCACAATTACCAACAACAAACCCTGATGCCCCAGTAATGTTGGATAGGATGTTAAGGTTATTAGCTTGCTATATTATATTGACATGCTCTGTAAGAACACAACAAGACGGTAAGGTACAAAGATTGTATGGATTAGCAACAGTAGCAAAATATTTAGTTAAGAATGAAGATGGTGTTTCTATGAGACGGCA | ||
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Performed PCA1 on CCOMT-1 | Performed PCA1 on CCOMT-1 | ||
''PCA Assembly'' | -'''PCA Assembly''' | ||
OK, so you've got a bunch of oligos, now what? First, use this recipe and program to do initial assembly of the oligos (do a separate one of these reactions for each chunk you're assembling): | -OK, so you've got a bunch of oligos, now what? First, use this recipe and program to do initial assembly of the oligos (do a separate one of these reactions for each chunk you're assembling): | ||
Recipe | Recipe | ||
38 uL ddH2O | 38 uL ddH2O | ||
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5 ul 2mM dNTPs | 5 ul 2mM dNTPs | ||
1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks) | 1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks) | ||
0.75 ul Expand polymerase | 0.75 ul Expand polymerase- | ||
From here, sample was given to John Wright: | From here, sample was given to John Wright: | ||
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Regular Zymo Cleanup on PCA1 -> PCA2 | Regular Zymo Cleanup on PCA1 -> PCA2 | ||
Regular Zymo Cleanup | <pre>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. | 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. | ||
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''DNA is now stuck to white filtered column'' | ''DNA is now stuck to white filtered column'' | ||
3) Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol) | 3) Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol) | ||
spin through, discard waste. | -spin through, discard waste. | ||
4) Add 200 uL of Zymo Wash Buffer | 4) Add 200 uL of Zymo Wash Buffer | ||
-spin through, discard waste. | -spin through, discard waste. | ||
5) spin for 90 seconds, full speed to dry. | 5) spin for 90 seconds, full speed to dry. | ||
6) elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction | 6) elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction</pre> | ||
Revision as of 10:16, 12 March 2013
2013 03 06: Designed oligos for PCA1 on CCOMT-1
CCOMT1_Synthon PCA for oligos 1-11, 13-14, 16 (407bp, PCA1_pdt) PCA for oligos 12,15, PCA1_pdt (462bp, PCA2_pdt) Digest PCA2_pdt (417+26+19, EcoRI/BamHI, PCA2_dig) Digest pBca9145-Bca1144 (2967+2958+9, EcoRI/BglII, Vector_dig) Ligate PCA2_dig and Vector_dig (2474, Bca1144-CCOMT1) CCOMT-1_oligo_12: ATATAGATGCCGTCCTAGCGAATTCATGAGATCTGCATCGTCTCATCGGTCTCCT CCOMT-1_oligo_1: TTATTTGTGTCTCACCTGTAGATCCCATAGGAGACCGATGAGACGATGCAGATCT CCOMT-1_oligo_9: ATGGGATCTACAGGTGAGACACAAATAACTCCTACACATATTTCTGATGAAGAAG CCOMT-1_oligo_3: GAGGCCAACTGCATTGCAAATAAGTTAGCTTCTTCATCAGAAATATGTGTAGGAG CCOMT-1_oligo_5: CTAACTTATTTGCAATGCAGTTGGCCTCTGCTTCAGTTTTGCCTATGATTTTGAA CCOMT-1_oligo_4: CTCTAATAAGTCCAATTCTAAAGCTGATTTCAAAATCATAGGCAAAACTGAAGCA CCOMT-1_oligo_11: ATCAGCTTTAGAATTGGACTTATTAGAGATTATTGCTAAGGCAGGTCCTGGAGC CCOMT-1_oligo_2: TGAGGCAATTTCGATAGGTGAAATTTGTGCTCCAGGACCTGCCTTAGCAATAAT CCOMT-1_oligo_14: ACAAATTTCACCTATCGAAATTGCCTCACAATTACCAACAACAAACCCTGATGC CCOMT-1_oligo_6: CCTTAACATCCTATCCAACATTACTGGGGCATCAGGGTTTGTTGTTGGTAATTG CCOMT-1_oligo_7: CCCAGTAATGTTGGATAGGATGTTAAGGTTATTAGCTTGCTATATTATATTGAC CCOMT-1_oligo_3: ACCGTCTTGTTGTGTTCTTACAGAGCATGTCAATATAATATAGCAAGCTAATAA CCOMT-1_oligo_10: ATGCTCTGTAAGAACACAACAAGACGGTAAGGTACAAAGATTGTATGGATTAGC CCOMT-1_oligo_8: ATTCTTAACTAAATATTTTGCTACTGTTGCTAATCCATACAATCTTTGTACCTT CCOMT-1_oligo_16: AACAGTAGCAAAATATTTAGTTAAGAATGAAGATGGTGTTTCTATGAGACGGCA CCOMT-1_oligo_15: AAGTATCTTTCCTGTGCCCAGGATCCATGCCGTCTCATAGAAACACCATCTTC
PCA1_pdt: AGATCTGCATCGTCTCATCGGTCTCCTATGGGATCTACAGGTGAGACACAAATAACTCCTACACATATTTCTGATGAAGAAGCTAACTTATTTGCAATGCAGTTGGCCTCTGCTTCAGTTTTGCCTATGATTTTGAAATCAGCTTTAGAATTGGACTTATTAGAGATTATTGCTAAGGCAGGTCCTGGAGCACAAATTTCACCTATCGAAATTGCCTCACAATTACCAACAACAAACCCTGATGCCCCAGTAATGTTGGATAGGATGTTAAGGTTATTAGCTTGCTATATTATATTGACATGCTCTGTAAGAACACAACAAGACGGTAAGGTACAAAGATTGTATGGATTAGCAACAGTAGCAAAATATTTAGTTAAGAATGAAGATGGTGTTTCTATGAGACGGCA PCA2_pdt: ATATAGATGCCGTCCTAGCGAATTCATGAGATCTGCATCGTCTCATCGGTCTCCTATGGGATCTACAGGTGAGACACAAATAACTCCTACACATATTTCTGATGAAGAAGCTAACTTATTTGCAATGCAGTTGGCCTCTGCTTCAGTTTTGCCTATGATTTTGAAATCAGCTTTAGAATTGGACTTATTAGAGATTATTGCTAAGGCAGGTCCTGGAGCACAAATTTCACCTATCGAAATTGCCTCACAATTACCAACAACAAACCCTGATGCCCCAGTAATGTTGGATAGGATGTTAAGGTTATTAGCTTGCTATATTATATTGACATGCTCTGTAAGAACACAACAAGACGGTAAGGTACAAAGATTGTATGGATTAGCAACAGTAGCAAAATATTTAGTTAAGAATGAAGATGGTGTTTCTATGAGACGGCATGGATCCTGGGCACAGGAAAGATACTT Bca1144-CCOMT1: gAATTCATGAGATCTGCATCGTCTCATCGGTCTCCTATGGGATCTACAGGTGAGACACAAATAACTCCTACACATATTTCTGATGAAGAAGCTAACTTATTTGCAATGCAGTTGGCCTCTGCTTCAGTTTTGCCTATGATTTTGAAATCAGCTTTAGAATTGGACTTATTAGAGATTATTGCTAAGGCAGGTCCTGGAGCACAAATTTCACCTATCGAAATTGCCTCACAATTACCAACAACAAACCCTGATGCCCCAGTAATGTTGGATAGGATGTTAAGGTTATTAGCTTGCTATATTATATTGACATGCTCTGTAAGAACACAACAAGACGGTAAGGTACAAAGATTGTATGGATTAGCAACAGTAGCAAAATATTTAGTTAAGAATGAAGATGGTGTTTCTATGAGACGGCATGGATCCtaaCTCGAGctgcaggcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccacaggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggatgatttctg
Will perform PCA1 on 2013-03-08
2013 03 08: Did not have PCR plates - will perform PCA1 on CCOMT-1 on 2013-03-09
2013 03 09: Performed PCA1 on CCOMT-1
-PCA Assembly -OK, so you've got a bunch of oligos, now what? First, use this recipe and program to do initial assembly of the oligos (do a separate one of these reactions for each chunk you're assembling): Recipe 38 uL ddH2O 5 ul 10x expand buffer 5 ul 2mM dNTPs 1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks) 0.75 ul Expand polymerase-
From here, sample was given to John Wright: Program (can run JCA/PCA1) 2 min initial denature at 94oC 30 sec denature at 94oC 30 sec anneal at 55oC [This should be the overlap temp of your oligos - vary as needed] 30 sec extension at 72oC repeat 2-4 30 times total 2013 03 12 Regular Zymo Cleanup on PCA1 -> PCA2
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. 1) Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction. ''ADB kills proteins and allows DNA to stick to the column'' 2) Transfer into the Zymo column (small clear guys) -spin through (1 minute, max g), discard waste. ''DNA is now stuck to white filtered column'' 3) Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol) -spin through, discard waste. 4) Add 200 uL of Zymo Wash Buffer -spin through, discard waste. 5) spin for 90 seconds, full speed to dry. 6) elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction
