SBB13Ntbk-LarryLi: Difference between revisions
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Sample was run in gel in lane 10. | Sample was run in gel in lane 10. | ||
Will perform zymo cleanup, digest and gel purify today. | Will perform zymo cleanup, digest and gel purify today. | ||
Heated and dissolved gel in ADP buffer and put in freezer. | |||
==[[User:Larry Li|Larry Li]] 12:54, 19 March 2013 (EDT)== | |||
Finished zymo cleaning up today. |
Revision as of 09:54, 19 March 2013
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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
Larry Li 12:42, 14 March 2013 (EDT)
Sample was run in gel in lane 10.
Will perform zymo cleanup, digest and gel purify today.
Heated and dissolved gel in ADP buffer and put in freezer.
Larry Li 12:54, 19 March 2013 (EDT)
Finished zymo cleaning up today.