SBB13Ntbk-LarryLi
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Larry Li 14:47, 5 March 2013 (EST)
CCOMT3 Construction File
Mix oligos (1-16) for one synthon, do PCA1 protocol (453 bp, pca1) PCR pca1 with external oligos (Newfoward and Newreverse) by PCA2 protocol (407 bp, pca2) Digest pca2 with EcoRI/BglII, gp (391+11+5 bp, pcr_dig) (get pre-digested vector from JCA of pBca9145-Bca1144) (EcoRI/BamHI, vect_dig) Ligate pcr_dig and vect_dig Oligo List: >CCOMT-3_oligo_1 TTTGGAATAGAAACAAACATGTCACCTCCGACATGTTGAGACGATGCAGATCTC >CCOMT-3_oligo_2 TTCCTTACCACCTGGATTGTGGGCTAACATGATTACGTCAATGTGGACTACAC >CCOMT-3_oligo_3 TTAGCCCACAATCCAGGTGGTAAGGAAAGAACACAGAAGGAGTTTGAAGACTT >CCOMT-3_oligo_4 GGCTTTGCCTGATAATGGTAAAGTTATTGTTGCCGAGTGCATTTTACCTGTCG >CCOMT-3_oligo_5 GGCCAAGGGTGCAGGTTTCCAGGGATTCAAGGTACATTGTAACGCATTTAATA >CCOMT-3_oligo_6 CTTTAGTTGCTAAAGATGAATCTGGTGCGACAGGTAAAATGCACTCGGCAACA >CCOMT-3_oligo_7 CTGTCATGATTGGTCTGACGAACATTGCTTGAAATTCTTAAAGAATTGTTATGA >CCOMT-3_oligo_8 CCTTCTTTAAGAACTCCATAATGTATGTATTAAATGCGTTACAATGTACCTTG >CCOMT-3_oligo_9 CATACATTATGGAGTTCTTAAAGAAGGTTGGATCCTGAGACCTGAGACGGCAT >CCOMT-3_oligo_10 CACCAGATTCATCTTTAGCAACTAAAGGTGTAGTCCACATTGACGTAATCATG >CCOMT-3_oligo_11 CAATGTTCGTCAGACCAATCATGACAGATCCATTTCATAAAGACGGCATCAGCC >CCOMT-3_oligo_12 ATATAGATGCCGTCCTAGCGAATTCATGAGATCTGCATCGTCTCAACATGTCGG >CCOMT-3_oligo_13 ATAACTTTACCATTATCAGGCAAAGCCTCATAACAATTCTTTAAGAATTTCAAG >CCOMT-3_oligo_14 AGGTGACATGTTTGTTTCTATTCCAAAGGCTGATGCCGTCTTTATGAAATGGAT >CCOMT-3_oligo_15 AATCCCTGGAAACCTGCACCCTTGGCCAAGTCTTCAAACTCCTTCTGTGTTCT >CCOMT-3_oligo_16 AAGTATCTTTCCTGTGCCCAGGATCCATGCCGTCTCAGGTCTCAGGATCCAA >CCOMT3-NewForward ccaaaGAATTCCGTCTCAACATGTCGGAGGTGAC >CCOMT3-NewReverse gactgAGATCTCGTCTCAGGTCTCAGGATCCAAC PCA1 Product: ATATAGATGCCGTCCTAGCGAATTCATGAGATCTGCATCGTCTCAACATGTCGGAGGTGACATGTTTGTTTCTATTCCAAAGGCTGATGCCGTCTTTATGAAATGGATCTGTCATGATTGGTCTGACGAACATTGCTTGAAATTCTTAAAGAATTGTTATGAGGCTTTGCCTGATAATGGTAAAGTTATTGTTGCCGAGTGCATTTTACCTGTCGCACCAGATTCATCTTTAGCAACTAAAGGTGTAGTCCACATTGACGTAATCATGTTAGCCCACAATCCAGGTGGTAAGGAAAGAACACAGAAGGAGTTTGAAGACTTGGCCAAGGGTGCAGGTTTCCAGGGATTCAAGGTACATTGTAACGCATTTAATACATACATTATGGAGTTCTTAAAGAAGGTTGGATCCTGAGACCTGAGACGGCATGGATCCTGGGCACAGGAAAGATACTT PCA2 Product: ccaaaGAATTCCGTCTCAACATGTCGGAGGTGACATGTTTGTTTCTATTCCAAAGGCTGATGCCGTCTTTATGAAATGGATCTGTCATGATTGGTCTGACGAACATTGCTTGAAATTCTTAAAGAATTGTTATGAGGCTTTGCCTGATAATGGTAAAGTTATTGTTGCCGAGTGCATTTTACCTGTCGCACCAGATTCATCTTTAGCAACTAAAGGTGTAGTCCACATTGACGTAATCATGTTAGCCCACAATCCAGGTGGTAAGGAAAGAACACAGAAGGAGTTTGAAGACTTGGCCAAGGGTGCAGGTTTCCAGGGATTCAAGGTACATTGTAACGCATTTAATACATACATTATGGAGTTCTTAAAGAAGGTTGGATCCTGAGACCTGAGACGAGATCTcagtc
On Thursday we will be doing the PCA.
Larry Li 13:06, 12 March 2013 (EDT)
On Thursday we performed the PCA1 protocol
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
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
Today we performed a zymo cleanup on the assembly reaction and will perform amplification
For the zymo cleanup we
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
and for the amplification
1 ul each outer oligo (10 uM)
1 ul purified pca product
.5 ul phusion
10 ul 5x phusion buffer
5 ul 2mM dNTPs
32.5 ul H2O
Program
2 min initial denature at 94oC
30 sec denature at 94oC
30 sec anneal at 60oC [This should be high, as your outer oligos now have a huge overlap with the correct product]
30 sec extension at 68oC
repeat 2-4 30 times total
