SBB13Ntbk-LarryLi

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