SBB09Ntbk-Simina Ticau: Difference between revisions
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- ladder: 3-5ul | - ladder: 3-5ul | ||
- run at 100V for about 20 minutes | - run at 100V for about 20 minutes | ||
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'''REGULAR ZYMO CLEANUP:''' | '''REGULAR ZYMO CLEANUP:''' | ||
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8. spin for 90 seconds, full speed to dry. | 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 | 9. elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction | ||
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'''Digest:''' | |||
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Revision as of 12:55, 11 February 2009
upaG Autotransporter short version
Hydroxyapatite-binding peptide
Beta Helix Repeats
Magnetic Bead Binding Peptide
~~*Simina Ticau 13:19, 11 February 2009 (EST):~~
Today we went through more protocols; I wrote down my notes here so I would have everything on one page for Monday.
OLIGOS :
- they will be at a concentration of 28.80 nmol and we want a final concentration of 288.0 ul --> add 288 ul of water (make sure you spin down the tubes before adding the water to make sure everything's at the bottom); mix well (tube rack) and spin down again --> oligo concentrations are now 100uM
PCR :
- first need to make an oligo dilution of: 9uL Water 1uL 100uM oligo You can throw away the remainder of the diluted oligo when you are done, but hold onto your stock tube! Set up the following reaction in a PCR tube (this is only for regular PCR, not Wobble Reaction): 24uL ddH2O 3.3uL 10x Expand Buffer "2" 3.3uL dNTPs (2mM in each) 1uL Oligo 1, 10uM 1uL Oligo 2, 10uM 0.5uL Expand polymerase "1" 0.5uL Template DNA
There are different programs: have to choose between C55, C2K55, C4K55, C8K55
If you get no product or poor yield of product, repeat the same PCR reaction as two different variants, both with the “45” program. The first reaction will be the same composition as the first. For the second one, don’t add 3.3 uL of the water and instead use the volume for 3.3 uL of DMSO (dimethylsulfoxide). If these PCRs fail as well, well you'd either redesign, use a different template source, or give up.
GEL:
- 5ul sample + 1ul dye - ladder: 3-5ul - run at 100V for about 20 minutes
REGULAR ZYMO CLEANUP:
The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction. It also will remove the buffer and restriction enzymes from a restriction digest reaction. 1. Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction (min. is 3 volumes) 2. Transfer into the Zymo column (small clear guys) 3. spin through (15 secs at max speed, ie around 15,000 rpm), discard waste. 4. Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol) 5. spin through, discard waste. 6. Add 200 uL of PE or 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
Digest:
~~*Simina Ticau 14:52, 9 February 2009 (EST):~~
- got the 2nd project ball
- made the construction files for Magnetic Bead BP & Beta Helix Repeats
- updated oligo page/construction file page/excel spreadsheet
Construction of Beta Helix Repeats basic part PCR Ost005F and Ost006R on E. coli avian APEC genomic DNA (703bp, EcoRI/BamHI) Sub into JCA_pBca9495AK-Bca1144 (EcoRI/BamHI, 3171+910, L) Product is JCA_pBca9495AK-M10042 {Beta Helix Repeats} ---------------------------- Ost005F Forward oligo for Beta Helix Repeats ccaaaGAATTCatgAGATCTagtgtcttcaacggcaccg Ost006R Reverse oligo for Beta Helix Repeats ctagcGGATCCtgtacgcatgacaaatgctgac
Wobble Ost007F/Ost008R (52 bp, EcoRI/BamHI) Sub into JCA_pBca9495AK-Bca1144 (EcoRI/BamHI, 3171+910, L) Product is JCA_pBca9495AK-M10043 {Magnetic Bead BP} ---- Ost007F Forward construction of Magnetic Bead BP basic part ccaaaGAATTCatgAGATCTCACCACAAATACTGGCACCG Ost008R Reverse construction of Magnetic Bead BP basic part ctagcGGATCCACGGTGCCAGTATTTGTGGTG
to do: Read articles
~~*Simina Ticau 14:20, 4 February 2009 (EST):~~
- got the project ball
- started working on the wiki & looking at the articles
- made the construction file for upaG & HydroxyapatiteBP
- updated oligo page/construction file page/excel spreadsheet
Construction of upaG basic part PCR Ost001F and Ost002R on CFT073 genomic DNA (298bp (282+16), EcoRI/BamHI) Sub into JCA_pBca9495KC-Bca1144 (EcoRI/BamHI, 2974+910, L) Product is JCA_pBca9495KC-M10026 {upaG} ---------------------------- Ost001F Cloning of upaG ccaaaGAATTCatgAGATCTgttgagatggataacaaactg Ost002R Cloning of upaG ctagcGGATCCttaCCACtgaataccggcaccgag
Wobble Ost003F/Ost004R (67 bp, EcoRI/BamHI) Sub into JCA_pBca9495AK-Bca1144 (EcoRI/BamHI, 3171+910, L) Product is JCA_pBca9495AK-M10027 {HydroxyapatiteBP} ---- Ost003F Forward construction of HydroxyapatiteBP basic part ccaaaGAATTCatgAGATCTgcgccgtggcatctgagcagccagtatag Ost004R Reverse construction of HydroxyapatiteBP basic part ctagcGGATCCggtgcggctatactggctgctcagatgc
to do: Read articles