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

ClassGel2

Lanes:

1	2	3	4	5	6	7	8	9	10
LZ AAAA PCA2	sbb1224	sbb1203	2-log ladder	AJ pca2	MD	JK caff-PCA2	AJ A	AJ B	PR PCR1

• 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

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

NheI/BamHI Digest for PCR product part SBB1202 and PCA2 product part SBB1230

NheI/BamHI Digest

For PCR product: 1. Digest PCR product by adding:

        8uL  of eluted PCR product
        1uL of NEB buffer 2
        0.5 uL of NheI
        0.5 uL of BamHI

2. incubate in thermocycler 37°C for 1 hour. 3. Run an agarose gel and cut out the band to minimize extra gel matter 4. Put in ependorf tube and add 600uL of Zymo ADB buffer (brown bottle) 5. heat at 55°C, shake and/or vortex until the gel has dissolved.

Note: I stopped here because time run out, the melted gel was kept frozen until next lab day

For PCA product:

1. Digest PCR product by adding:

        8uL  of eluted PCR product
        1uL of NEB buffer 2
        0.5 uL of NheI
        0.5 uL of BamHI

2. incubate in 37°C for 1 hour.

3. Proceed to another Zymo small fragment cleanup

Note: I did not use wobble for this digestion, therefore ligation might not work

Pamela Rios 14:23, 28 February 2012 (EST)

Part: SBB1202

Continuing cleaning up gel fragment:

1. transfer melted gel solution into the Zymo column inside a collection tube (small clear guys) 2. spin through (15 sec), discard waste. 3. Add 200 uL of Zymo Wash Bbuffer (which is basically 70% ethanol) 4. spin through (15 sec), discard waste. 5. Add 200 uL of Zymo Wash Buffer 6. spin through (15 sec), discard waste. 7. spin for 90 seconds, full speed to dry. 8. elute with water 8uL and spin for 60 seconds into a fresh Eppendorf tube and store on ice

Part: SBB1202 Part: SBB1230

Performed a Ligation and transformation for both parts on Vector PBca9525-Bca1834*

Ligation of NheI/BamHI digests

1.Set up the following reaction

         6.5uL ddH2O
         1uL T4 DNA Ligase Buffer 
         1uL Vector digest *
         1uL Insert digest 
         0.5uL T4 DNA Ligase

2.Pound upside down on the bench to mix 3.Give it a quick spin to send it back to the bottom of the tube 4.Incubate on the benchtop for 30min 5.Put on ice and proceed to the transformation

Transformation by heat-shock

 1. Thaw a 200 uL aliquot of cells on ice
 2. Add 50 uL of water to the cells
 3. Add 30 uL of KCM to the cells 
 4. Put my ligation mixtures on ice, let cool a minute or two 
 5. Add 70 uL of the cell cocktail to the ligation,   stir to mix
 6. Let sit on ice for 10 min
 7. Heat shock for 90 seconds at 42 (longer incubation may work better)
 8. Put back on ice for 1 min
 9. Add 100uL of 2YT, let shake in the 37 degree incubator for 45 minutes
11. plate 75 uL on selective antibiotics 
12. incubate at 37 degrees UPSIDE DOWN overnight**

note: did not add water, also I did a negative control by adding water instead of the ligation.

(*) digestion of vector PBca9525-Bca1834 with NheI/BamHI was done by GSI (**) GSI will freeze

Pamela Rios 14:23, 1 March 2012 (EST)

Part: SBB1202 Colony: #1, #2 Part: SBB1230 Colony: #1

Miniprep purification of DNA

  1. Pellet 2 mL saturated culture Tar by spinning full speed, 30 seconds in a 2mL Microcentrifuge tube.
  2. Dump supernatant
  3. Add 250uL of P1 buffer into each tube. Resuspend the cells thoroughly
  4. Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution   
      should become clearer.
  5. Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to  
      precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
  6. Spin in centrifuge at top speed for 5 minutes.
  7. Label blue columns with an alcohol-resistant lab pen.
  9. Pour liquid into columns, and place the columns into the centrifuge. Spin at full speed for 15 seconds.
  10. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
  11. Wash each column with 500 uL of PB buffer.
  12. Spin in centrifuge at full speed for 15 seconds, then flick out the liquid again.
  13. Wash with 750uL of PE buffer (washes the salts off the resins).
  14. Spin in centrifuge at full speed for 15 seconds and flick out liquid again.
  15. Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol.
  16. Label new Microcentrifuge tubes and put columns in them.
  17. Elute them by adding 50uL of water down the middle of the column (don't let it stick to the sides).
  18. Spin in centrifuge at top speed for 30 seconds.
  19.Take out columns and cap the tubes and store on the ice for mapping.

Pick up colonies and inoculate LB liquid medium for:

Part: SBB1202 Colony: #3, #4 Part: SBB1230 Colony: #2, #3, #4

Picking of colonies

  Add 4mL of LB media with the appropriate antibiotics to a clean test tube
  Pick a well-isolated, round, and "normal" looking colony with a toothpick
  Drop it in the test tube
  Incubate at 37 overnight

Pamela Rios 14:23, 2 March 2012 (EST)

Analytical Digest for

Part: SBB1230 Colony: #1

 1. Set up the following 10uL reaction in a PCR tube and mix well:
                 6uL ddH2O                  
                 2uL 0.1X Miniprepped plasmid of pBca9525-sbb1230 (lz_IILK)
                 1uL 10x NEB Buffer 2
                 0.5uL NheI
                 0.5uL Xhol 
  2. Incubate at 37°C on the thermocycler for 30 minutes

Part: SBB1202 Colony: #1, #2

 1. Set up the following 10uL reaction in a PCR tube and mix well:
                 6uL ddH2O                  
                 2uL 0.1X Miniprepped plasmid of pBca9525-sbb1230 (lz_IILK)
                 1uL 10x NEB Buffer 2
                 0.5uL EcoRI
                 0.5uL Xhol 
  2. Incubate at 37°C on the thermocycler for 30 minutes

I added 4ul of gel dye for each digest and freeze samples because I run out of time

Miniprep purification DNA for:

Part: SBB1202 Colony: #3, #4 Part: SBB1230 Colony: #2, #3, #4

Pamela Rios 14:23, 6 March 2012 (EST)

Run gel for Analytical Digest for

Part: SBB1202 Colony: #1, #2 Part: SBB1230 Colony: #1

Class Gel 2

Lanes:

1	2	3	4	5	6	7	8	9	10
AJ A+B	JS 1201-1	JS 1201-2	JS 1226-1	JS 1226-2	Ladder	PR 02-1	PR 02-2	PR 30-1	JK 1233-1

I re-digested Part: SBB1202 Colony: #1, #2 along with colony: #3, #4 with a mix of 3 restriction enzyme that could help distinguish parent from product plasmid due to insert and target sequence where similar in length. Previous ( March 2) digestion of the product pBca9525-sbb1230 might not have been sufficient enough to asses distinguish parent from product plasmid.

Part: SBB1202 Colony: #1, #2,#3,#4

 1. Set up the following 10uL reaction in a PCR tube and mix well:
                 6uL ddH2O                  
                 2uL 0.1X Miniprepped plasmid of pBca9525-sbb1202 (araC)
                 1uL 10x NEB Buffer 2
                 0.5uL NheI
                 0.5uL EcoRV 
                 0.5uL BamHI
 2. Incubate at 37°C on the thermocycler for 30 minutes

Part: SBB1230 Colony: #2, #3, #4

 1. Set up the following 10uL reaction in a PCR tube and mix well:
                 6uL ddH2O                  
                 2uL 0.1X Miniprepped plasmid of pBca9525-sbb1230 (lz_IILK)
                 1uL 10x NEB Buffer 2
                 0.5uL EcoRI
                 0.5uL Xhol 
  2. Incubate at 37°C on the thermocycler for 30 minutes

Gel Results

Class Gel 4

Lanes:

1	2	3	4	5	6	7	8	9	10
MM 1232-1	MM 1232-2	MM 1215-2	DC 1221 A+ B Pur	Ladder	JK 1233-2	JK 1204-1	JK 1204-2	PR 30-2	PR 30-3

Class Gel 5

Lanes:

1	2	3	4	5	6	7	8	9	10
PR 30-4	PR 02-1	PR 02-2	PR 02-3	PR 02-4	Ladder	JK 1223-1	AJ 12205-1	AJ 12205-2	AJ 12205-3

Pamela Rios 14:23, 8 March 2012 (EST)

Sequence