SBB12Ntbk-Pamela Rios

Pamela Rios 16:23, 13 February 2012 (EST)

This is my next entry.

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Pamela Rios 14:23, 17 February 2012 (EST)

Part: SBB1202

Oligos: osbb1202F , osbb1202R Template: pBAD-T4L pcrpdt:916bp

1. Prepare 10 uM solution for each oligo

9uL Water 1uL 100uM oligo

2. Set up PCR (clonning)

24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL Oligo ca998, 10uM
1uL Oligo osbb1333R, 10uM
0.5uL Expand polymerase "1"
0.5uL Template DNA

3. Store in ice

• PCR program run by GSI

Part: SBB1230

oligos: o1, o2, o10

1. Mix 1uL of each oligo (100uM)

2. Set up PCA (step1) for "lz_IILK"

    38 uL ddH2O
    5 ul 10x expand buffer
    5 ul 2mM dNTPs
    1 ul oligo mixture (100uM total, mixture of oligos from step 1)
    0.75 ul Expand polymerase

3. store on ice

• PCA program run by GSI

      2 min initial denature at 94oC
     30 sec denature at 94oC
     30 sec anneal at 55oC [This should be the overlap temp of your oligos - vary as needed]
     30 sec extension at 72oC
     repeat 2-4 30 times total

Pamela Rios 14:23, 21 February 2012 (EST)

Part: SBB1202

PCR product analysis 

1. Mix 6uL Tar PCR product AraC and 4uL dye, then load into the well#10 of gel*#2

2.run the gel for about 30 mins, then take a picture.

3.Cut product band to be Zymo purified

 cut out bands minimizing extra gel matter.
 put in ependorf tube and add 600uL of Zymo ADB buffer (brown bottle).
 heat at 55, shake and/or vortex until the gel has dissolved.
 If the DNA is <300bp add 250uL of isopropanol (mine was above 900bp)
 transfer into the Zymo column inside a collection tube (small clear guys)
 spin 15 sec through, discard waste.
 Add 200 uL of Zymo Wash Bbuffer (which is basically 70% ethanol)
 spin  15 sec through, discard waste.
 Add 200 uL of Zymo Wash Buffer
 spin 15 sec through, discard waste.
 spin for 90 seconds, full speed to dry.
 elute with water into a fresh Eppendorf tube ( I used 8ul)

4.Store the rest of PCR product in freezer

5. Store the purified gel product

• Performed by GSI

Part: SBB1230 (PCA2)

oligos: o1, o2

2. Set up PCA (step2) for "lz_IILK PCA1"

 Add 100 uL of Zymo ADB buffer (brown bottle) to the PCA1 tube.
 Transfer all liquid from PCA1 tube into the Zymo column 
 Add 500uL of Ethanol and pipette up and down to mix
 spin through 15 seconds, discard waste.
 Add 200 uL of Zymo Wash Buffer (white bottle which is basically 70% ethanol)
 spin through 15 seconds, discard waste.
 Add 200 uL of Zymo Wash Buffer
 spin through 15 seconds, discard waste.
 spin for 90 seconds, full speed to dry.
 add water and elute with water (spin 60s) into a fresh Eppendorf tube labeled (I used 50ul)
 

3. Skip running a gel

4. Set up amplification "PCA2 lz_IILK"

              32.5 ul H2O
              5 ul 2mM dNTPs
              10 ul 5x phusion buffer
              1 ul purified PCA1 product (from step 2)
              1 ul  outer oligo o1 (10 uM)
              1 ul  outer oligo o12 (10 uM)
              0.5 ul phusion

5. Store on ice

• PCA2 program run by GSI

   2 min initial denature at 94°C
   30 sec denature at 94°C
   30 sec anneal at 60°C [This should be high, as your outer oligos now have a huge overlap with the correct product]
   30 sec extension at 68°C
   repeat 2-4 30 times total