SBB13Ntbk-Harini Sadeeshkumar

Vanillin - quiC-3

PCA quiC-3_oligo_1-16                                   (460 bp, pca1)
PCR pca1 with quiC-3_oligo_11/quiC-3_oligo_15           (460 bp, pca2)
Digest pca2, gp                                         (EcoRI/BamHI, 20+415+25 bp, pcr_dig)
(get pre-digested vector from JCA of pBca9145-Bca1144   (EcoRI/BamHI, vect_dig)
Ligate pcr_dig and vect_dig			        (pBca9145-quiC-3)

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<quiC-3_oligo_1
TGATCTAATTGCAATTCCAATCCTGTTTCCATGAACCTTATTTGAATCGAA

<quiC-3_oligo_2
TCGTAGGTAATAAGTTCGAGAACTATTCTGACGGTTTGCAAATCAATTGGG

<quiC-3_oligo_3
TCCTTCGTGTTCCAAAGCTGGAACACCGAAAACGATTGCACCTAATCTTTG

<quiC-4_oligo_4
TCAATTTAACAGGAGATGGTAACATTTTCGATTCAAATAAGGTTCATGGAA

<quiC-5_oligo_5
GTGTTCCAGCTTTGGAACACGAAGGATTTGTAGGTTCTATGAGACGGCATG

<quiC-6_oligo_6
GTAAAAGAAACTACATTGCTTATAATGAATTGACAAATAACTCTTTGGGTT

<quiC-7_oligo_7
CATTAACTAAGAACCAAATTTGGGATAATGGAAAGGACATCAAGAGGTGTG

<quiC-8_oligo_8
ATTTGGAACACAAGAACCTCCAGCTTCACACCTCTTGATGTCCTTTCCATT

<quiC-9_oligo_9
ATTATAAGCAATGTAGTTTCTTTTACCCCAATTGATTTGCAAACCGTCAGA

<quiC-10_oligo_10
ATCCCAAATTTGGTTCTTAGTTAATGTGATTCTTGCATTAGCATCCTTTTC

<quiC-11_oligo_11
ATATAGATGCCGTCCTAGCGAATTCATGAGATCTGCATCGTCTCAAATGCTG

<quiC-12_oligo_12
ATAGTTCTCGAACTTATTACCTACGACAGCATTTGAGACGATGCAGATCTCA

<quiC-13_oligo_13
ACAGGATTGGAATTGCAATTAGATCAGAAAAGGATGCTAATGCAAGAATCA

<quiC-14_oligo_14
AATGTTACCATCTCCTGTTAAATTGAAACCCAAAGAGTTATTTGTCAATTC

<quiC-15_oligo_15
AAGTATCTTTCCTGTGCCCAGGATCCATGCCGTCTCATAGAACCTACAAA

<quiC-16_oligo_16
AAGCTGGAGGTTCTTGTGTTCCAAATCAAAGATTAGGTGCAATCGTTTTCG

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pca1/pca2:
ATATAGATGCCGTCCTAGCGAATTCATGAGATCTGCATCGTCTCAAATGCTGTGAGATCTGCATCGTCTCAAATGCTGTCGTAGGTAATAAGTTCGAGAACTATTCTGACGGTTTGCAAATCAATTGGGGTAAAAGAAACTACATTGCTTATAATGAATTGACAAATAACTCTTTGGGTTTCAATTTAACAGGAGATGGTAACATTTTCGATTCAAATAAGGTTCATGGAAACAGGATTGGAATTGCAATTAGATCAGAAAAGGATGCTAATGCAAGAATCACATTAACTAAGAACCAAATTTGGGATAATGGAAAGGACATCAAGAGGTGTGAAGCTGGAGGTTCTTGTGTTCCAAATCAAAGATTAGGTGCAATCGTTTTCGGTGTTCCAGCTTTGGAACACGAAGGATTTGTAGGTTCTATGAGACGGCATGGATCCTGGGCACAGGAAAGATACTT


3/8/13 -- Assembly Step:
Assembly:
1. 38 uL ddH2O
2. 5 ul 10x expand buffer
3. 5 ul 2mM dNTPs
4. 1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks)
5. 0.75 ul Expand polymerase
Program (can run JCA/PCA1)
1. 2 min initial denature at 94oC
2. 30 sec denature at 94oC
3. 30 sec anneal at 55oC [This should be the overlap temp of your oligos - vary as needed]
4. 30 sec extension at 72oC
5. repeat 2-4 30 times total

3/12/13 -- Zymo Cleanup and Amplification
Zymo Cleanup:
1. Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
2. Transfer into the Zymo column (small clear guys)
3. spin through, discard waste.
4. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
5. spin through, discard waste.
6. Add 200 uL of Zymo Wash Buffer
7. spin through, discard waste.
8. spin for 90 seconds, full speed to dry.
9. elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction

Amplification:
1. 1 ul each outer oligo (10 uM)
2. 1 ul purified pca product
3. .5 ul phusion
4. 10 ul 5x phusion buffer
5. 5 ul 2mM dNTPs
6. 32.5 ul H2O
Program
1. 2 min initial denature at 94oC
2. 30 sec denature at 94oC
3. 30 sec anneal at 60oC [This should be high, as your outer oligos now have a huge overlap with the correct product]
4. 30 sec extension at 68oC
5. repeat 2-4 30 times total

3/14/13- Zymo Cleanup, Digestion, Run on gel
Digestion:
1. 8uL of eluted PCR product
2. 1uL of NEB Buffer 2
3. 0.5uL EcoRI
4. 0.5uL BamHI
5. Incubate at 37 degrees on the thermocycler for 1hr

Run on gel
cut out band
begin gel purification--stop after gel is dissolved in ADB buffer

3/19/13- Finish gel cleanup
gel purification:
1. cut out bands minimizing extra gel matter.
2. put in ependorf tube and add 600uL of Zymo ADB buffer (brown bottle).
3. heat at 55, shake and/or vortex until the gel has dissolved.
4. If the DNA is <300bp add 250uL of isopropanol
5. transfer into the Zymo column inside a collection tube (small clear guys)
6. spin through, discard waste.
7. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
8. spin through, discard waste.
9. Add 200 uL of Zymo Wash Buffer
10. spin through, discard waste.
11. spin for 90 seconds, full speed to dry.
12. elute with water into a fresh Eppendorf tube