SBB09Ntbk-Simina Ticau

upaG Autotransporter short version
Hydroxyapatite-binding peptide
Magnetic Bead Binding Peptide
Beta Helix Repeats

~~*simina_ticau@berkeley.edu 14:12, 18 February 2009 (EST):~~

Today I set up the Wobble reactions for Hydroxyapatite-binding peptide and Magnetic Bead Binding Peptide and the PCR reaction for upaG Autotransporter short version. (I'm no longer working on the Beta Helix Repeats)

~~*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 (may be at a diff concentration, need to check) and we want a final concentration
of 100uM --> add 288 ul of water (if 28.80 nmol)
(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:

For PCR products, you will only digest a portion of your purified PCR product.  Note that you must make a minor modification to the procedure if your DNA is shorter than 300bp (you add a little isopropanol to the ADB after melting)
*Set up the following reaction:
  8uL of eluted PCR product
  1uL of NEB Buffer 2
  0.5uL EcoRI
  0.5uL BamHI
*Incubate at 37 degrees on the thermocycler for 1hr
*Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees.  ****NOTE:  If you are running short of time, this is an acceptable stopping point
*If the DNA is shorter than 300bp, add 250uL of isopropanol and mix prior to loading it on the column

~~*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