SBB12Ntbk-GregFukushima: Difference between revisions
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*Cut out the bands and melt with 600uL of ADB buffer at 55 degrees. | *Cut out the bands and melt with 600uL of ADB buffer at 55 degrees. | ||
*Freeze and save for next time. | *Freeze and save for next time. | ||
==[[User:Greg_Fukushima|Greg Fukushima]], 8 March 2012== | |||
- redid the PCA gel because the one from feb 24 was not labeled | |||
- also set up the PCR for A+B to make pcrpdt for SOEing |
Revision as of 12:03, 8 March 2012
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 oligo dilutions of ca998/gfRevPR:
9uL Water 1uL 100uM oligo ca998 or gfRevPR
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:
- 38 uL ddH2O
- 5 ul 10x expand buffer
- 5 ul 2mM dNTPs
- 1 ul oligo mixture(o1,o10,o12) (100uM total, mixture of oligos after combination of 100uM stocks)
- 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. Also did amplification step of the Leucine Zipper PCA reaction. Finally did the Small-Frag Zymo Cleanup on "A" from the SOEing PCR construction file.
Protocol for PCR:
Made oligo dilutions of gfForRbsCmR/gfRevRbsCm:
9uL Water 1uL 100uM oligo gfForRbsCmR or gfRevRbsCm
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.
Protocol for PCA:
Added following into PCR tube for the PCA reaction:
- 1 ul of each 10ul outer oligo(o1/o12)
- 1 ul purified pca product(PCA1)
- .5 ul phusion
- 10 ul 5x phusion buffer
- 5 ul 2mM dNTPs
- 32.5 ul H2O
Actual reaction was done by Zach.
Small-Frag Zymo Cleanup Protocol:
- Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction product "A".
- Transfer into the Zymo column
- Add 500uL of Ethanol and pipette up and down to mix
- spin through (15s), discard waste.
- Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
- spin through (15s), discard waste.
- Add 200 uL of Zymo Wash Buffer
- spin through, discard waste.
- spin for 90 seconds, full speed to dry.
- elute with water (spin 60s) into a fresh Eppendorf tube
Greg Fukushima, 23 February 2012
Did partial EcoRI/BamHI digest of PCA product as well as the gel for the 2 PCR reactions from before.
Protocol for Gels:
- For each PCR, load 6uL of PCR product premixed with 4uL of loading buffer in a single well of a 1% agarose gel.
- Run gel
- Cut out the bands, put them into a single 1.5mL microcentrifuge tube.
- Add 650uL of ADB buffer
- Freeze to store for tomorrow.
My lanes were lanes 1 and 2. Lane 1 was pKGC438 and lane 2 was pBjk2741-jtk2801.
NheI/BamHI Digest of PCA Product:
- Set up the following reaction:
8uL of eluted PCA product 1uL of NEB Buffer 2 0.5uL NheI 0.5uL BamHI
- Incubate at 37 degrees on the thermocycler for 1hr
- Store for gel tomorrow
Greg Fukushima, 24 February 2012
Ran the Zymo Gel Purification on the gel PCR products from yesterday. Also did the gel for the PCA NheI/BamHI Digest from yesterday.
Protocol for Zymo Gel Purification:
- All spins until the drying step are 15 second full speed spins.
- heat the bands + ADB buffer in ependorf tube at 55, shake and/or vortex until the gel has dissolved.
- If the DNA is <300bp add 250uL of isopropanol
- transfer into the Zymo column inside a collection tube (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
Protocol for continuation of NheI/BamHI digest of PCA:
- Mix the digested PCA product with 2uL of loading buffer.
- Put mixture into the gel and run.
- Cut out the bands and melt with 600uL of ADB buffer at 55 degrees.
- Freeze and save for next time.
Greg Fukushima, 8 March 2012
- redid the PCA gel because the one from feb 24 was not labeled - also set up the PCR for A+B to make pcrpdt for SOEing