SBB09Ntbk-Simina Ticau

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

WOBBLE REACTION:

The Wobble procedure is a variation of Klenow Extension that begins with two oligonucleotides that overlap by around 20 bp on
their 3' ends and uses a thermostable polymerase

*Order the oligos, they don't need to be purified in any special way, smallest scale is ok
*Make 100uM stocks, this is the concentration used directly for the reaction
*Prepare the following reaction:
  29 uL water
  5 uL Expand 10x Buffer 2
  5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
  5 uL Oligo 1 (100uM)
  5 uL Oligo 2 (100uM)
  0.75 uL Expand Polymerase 1

*Run the wobble program, whick is:
  2 min at 94
  ''10 cycles of:''
  30 sec at 55
  30 sec at 72
  (or something similar)
*There is no point in running an analytical gel afterwards, there is nothing to see
*You'll want to run short fragment cleanups to remove the polymerase prior to digestion steps

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

~~*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