SBB12Ntbk-GregFukushima

Greg Fukushima, 7 February 2012

This is my first day of notebooks for 140L! I love wikis!

Greg Fukushima, 16 February 2012

First Day of PCR and cloning:

Construction File for SOEing PCR

PCR ca998/gfRevPR on pBjk2741-jtk2801	    	(160bp, A)
PCR gfForRbsCmR/gfRevRbsCmR on pKGC438    	(920bp, B)
PCR ca998/gfRevRbsCmR on A+B         		(1059bp, pcrpdt)
Digest pcrpdt                    		(EcoRI/BamHI, L, pcrdig)
Digest pBgl00001-Bjk2828         		(EcoRI/BamHI, L, plasdig)
Ligate pcrdig and plasdig, product is pBgl00001-sbb1220
----
>ca998	Forward external annealing for purification of P_R part
gtatcacgaggcagaatttcag
>gfForRbsCmR
ggtgataatggttgcGGATCTCCACAACGGTTTCCCTC
>gfRevRbsCmR
gctagGGATCCTTACGCCCCGCCCTGCCAC
>gfRevPR
AGATCCgcaaccattatcacc
>pcrpdt
GTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATCTGAAGTGGAATTCATGAGATCTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGCGGATCTCCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGAGCGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACGATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAGGATCCCTAGC

Protocol for PCR:

Made an oligo dilution of:

 9uL Water
 1uL 100uM oligo

Set up the following reaction in a PCR tube:

24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL gfRevPR, 10uM
1uL ca998, 10uM
0.5uL Expand polymerase "1"
0.5uL pBjk2741-jtk2801 Template DNA

Actual reaction was done by Zach.

NOTE: I was only able to do the first PCR reaction on my construction file because pKGC438 was not available.

Construction File for Leucine Zipper PCA

PCA1 on o1,o10,o12        (pca1)
PCA2 with o1/o12 on pca1  (139 bp, pca2)
Digest pca2               (NheI/BamHI, L, 1210dig)
Digest pBca9525-Bca1834   (NheI/BamHI, L, vectdig)
Ligate 1210dig + vectdig, product is pBca9525-sbb1210
----
>o1	
CCATAgctagcGGCAGTGGATCTGTTAAAGAACTGGAAGACAAAAACGAAGAACTGCTGAGT
>o10
CAAAAACGAAGAACTGCTGAGTATCATCTACCACCTGAAAAACGAAGTTGCTCGTCTGA
>o12
CAGTAGGATCCTTAGCCGCCACGTTCGCCAACCAGTTTTTTCAGACGAGCAACTTCGTT
>pca2
CCATAgctagcGGCAGTGGATCTGTTAAAGAACTGGAAGACAAAAACGAAGAACTGCTGAGTATCATCTACCACCTGAAAAACGAAGTTGCTCGTCTGAAAAAACTGGTTGGCGAACGTGGCGGCTAAGGATCCTACTG

Protocol for PCA:

Added following into PCR tube for the PCA reaction:

1. 38 uL ddH2O
2. 5 ul 10x expand buffer
3. 5 ul 2mM dNTPs
4. 1 ul oligo mixture(o1,o10,o12) (100uM total, mixture of oligos after combination of 100uM stocks)
5. 0.75 ul Expand polymerase

Actual reaction was done by Zach.

Greg Fukushima, 17 February 2012

Continued PCR and PCA reactions. Did the second PCR reaction listed on the construction file.

Protocol for PCR:

Made an oligo dilution of:

 9uL Water
 1uL 100uM oligo

Set up the following reaction in a PCR tube:

24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL gfRevRbsCmR, 10uM
1uL gfForRbsCmR, 10uM
0.5uL Expand polymerase "1"
0.5uL  pKGC438 Template DNA

Actual reaction was done by Zach.

Also did amplification step of the Leucine Zipper PCA reaction.

Protocol for PCA:

Added following into PCR tube for the PCA reaction:

1. 1 ul of each 10ul outer oligo(o1/o12)
2. 1 ul purified pca product(PCA1)
3. .5 ul phusion
4. 10 ul 5x phusion buffer
5. 5 ul 2mM dNTPs
6. 32.5 ul H2O

Actual reaction was done by Zach.