SBB13Ntbk-Tai Lun Ng: Difference between revisions
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1. Make a 100uM 1 ul oligo mixture of FaCCOMT-2_oligo_1 to FaCCOMT-2_oligo_16 <br/> | 1. Make a 100uM 1 ul oligo mixture of FaCCOMT-2_oligo_1 to FaCCOMT-2_oligo_16 <br/> | ||
2. Set up the PCA reaction by mixing: <br/> | 2. Set up the PCA reaction by mixing: <br/><br/> | ||
38 uL ddH2O<br/> | 38 uL ddH2O<br/> | ||
5 ul 10x expand buffer<br/> | 5 ul 10x expand buffer<br/> | ||
5 ul 2mM dNTPs<br/> | 5 ul 2mM dNTPs<br/> | ||
1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks)<br/> | 1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks)<br/> | ||
0.75 ul Expand polymerase<br/> | 0.75 ul Expand polymerase<br/><br/> | ||
3. | 3.Run on the PCR machine this program:<br/>br/> | ||
Program (can run JCA/PCA1)<br/> | |||
2 min initial denature at 94oC<br/> | |||
30 sec denature at 94oC<br/> | |||
30 sec anneal at 55oC [This should be the overlap temp of your oligos - vary as needed]<br/> | |||
30 sec extension at 72oC<br/> | |||
repeat 2-4 30 times total<br/><br/> | |||
4. The expected product is :<br/> | |||
ATATAGATGCCGTCCTAGCGAATTCATGAGATCTGCATCGTCTCAGATGGTGTTTCTATCGCAGCATTGTGCTTAATGAACCAAGATAAGGTCTTAGTCGAGTCATGGTATCATTTGAAAGATGCTGTTTTGGACGGTGGTATTCCTTTTAATAAGGCATACGGAATGACAGCCTTCGATTACCATGGTACTGATCCAAGATTTAATAAAGTTTTCAACAAAGGAATGGCTGACCACTCTACTATTACAATGAAGAAAATCTTGGAAACTTACAAGGGTTTCGAGGGTTTAAAATCAATAGTAGACGTAGGTGGTGGAACTGGTGCTGTTGTTAATATGATTGTATCTAAATATCCTTCAATTAAGGGTATAAATTTCGACTTACCTCATGTAATTGAGGATGCTCCACAATACCCAGGTGTCCAGCATGAGACGGCATGGATCCTGGGCACAGGAAAGATACTT (465bp)<br/><br/> | |||
5. Run a zymo cleanup: | |||
Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.<br/> | |||
Transfer into the Zymo column (small clear guys)<br/> | |||
spin through, discard waste.<br/> | |||
Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)<br/> | |||
spin through, discard waste.<br/> | |||
Add 200 uL of Zymo Wash Buffer<br/> | |||
spin through, discard waste.<br/> | |||
spin for 90 seconds, full speed to dry.<br/> | |||
elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction<br/><br/> | |||
6. Set up another PCR reaction with FaCCOMT-2_oligo_12 and FaCCOMT-2_oligo_14. | |||
Revision as of 13:15, 5 March 2013
March 5, 2013
Construction File for PCA assembly of FaCCOMT-2
1. Make a 100uM 1 ul oligo mixture of FaCCOMT-2_oligo_1 to FaCCOMT-2_oligo_16
2. Set up the PCA reaction by mixing:
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
3.Run on the PCR machine this program:
br/>
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
4. The expected product is :
ATATAGATGCCGTCCTAGCGAATTCATGAGATCTGCATCGTCTCAGATGGTGTTTCTATCGCAGCATTGTGCTTAATGAACCAAGATAAGGTCTTAGTCGAGTCATGGTATCATTTGAAAGATGCTGTTTTGGACGGTGGTATTCCTTTTAATAAGGCATACGGAATGACAGCCTTCGATTACCATGGTACTGATCCAAGATTTAATAAAGTTTTCAACAAAGGAATGGCTGACCACTCTACTATTACAATGAAGAAAATCTTGGAAACTTACAAGGGTTTCGAGGGTTTAAAATCAATAGTAGACGTAGGTGGTGGAACTGGTGCTGTTGTTAATATGATTGTATCTAAATATCCTTCAATTAAGGGTATAAATTTCGACTTACCTCATGTAATTGAGGATGCTCCACAATACCCAGGTGTCCAGCATGAGACGGCATGGATCCTGGGCACAGGAAAGATACTT (465bp)
5. Run a zymo cleanup:
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
6. Set up another PCR reaction with FaCCOMT-2_oligo_12 and FaCCOMT-2_oligo_14.
