SBB12Ntbk-Huiting He

Huiting He 14:25, 16 February 2012 (EST)

==PCR product of Tar and PCA1 of Lz-AAAA-1==


===PCR product of Tar===
1. Obtain my oligos hhe0120F(32.6nM) and hhe0120R(32.9nM), dilute each oligo into 100uM by adding 326uL and 329uL ddH2O, respectively. <br>
2. Prepare 10uM of each oligo dilution by mixing
        9uL Water
        1uL 100uM oligo
3. label PCR tube "Tar", and add
        24uL ddH2O
        3.3uL 10x Expand Buffer "2"
        3.3uL dNTPs (2mM in each)
        1uL Oligo hhe0120F, 10uM
        1uL Oligo HHE0120R, 10uM
        0.5uL Template DNA "MG1655 Gen."
        0.5uL Expand polymerase "1"  (lastly)
4. Mix well and centrifuge briefly, then store on ice.

***The next steps (PCR amplification) are running by GSI:
     Note:first 10 cycles have shorter extensions than tha latter 20 cycles.
     Since the size of my predicted product is 802bp, which is under 2kb, we should use 2K55


===PCA1 product of Lz-AAAA-1===
1. label tube "Mix" and add 1uL of each oligo o1, o11, o12, then mix it well and centrifuge.<br>
2. label PCR tube "Lz-AAAA-1 PCA1", and add
     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. Mix well and centrifuge briefly, then store on ice.

***The next steps (JCA/PCA1 Program) are running by GSI:
      a) 2 min initial denature at 94°C
      b) 30 sec denature at 94°C
      c) 30 sec anneal at 55°C [This should be the overlap temp of your oligos - vary as needed]
      d) 30 sec extension at 72°C
      e)repeat 2-4 30 times total

Huiting He 00:53, 18 February 2012 (EST)

==Analytical gel of PCR product(Tar) and PCA2 of Lz-AAAA-1==


===Analytical gel of PCR product(Tar)===
1.Obtain my PCR product Tar
2.label tube "tar-gel", mix 6uL Tar PCR product Tar and 4uL dye, then load into the well(#8) of gel1 prepared by GSI. <br>
3.run the gel for about 30 mins, then take a picture.
4.Save the PCR product of Tar in the freezer for later.


===PCA2 of Lz-AAAA-1===
1. Obtain PCA1 product.
2. Since the product of PCA1 is expected small fragment (less than 300bp0, use small-frag Zymo cleanup procedure:
      a) Add 100 uL of Zymo ADB buffer (brown bottle) to the PCA1 tube.
      b)Transfer all liquid from PCA1 tube into the Zymo column 
      c)Add 500uL of Ethanol and pipette up and down to mix
      d)spin through 15 seconds, discard waste.
      e)Add 200 uL of Zymo Wash Buffer (white bottle which is basically 70% ethanol)
      f)spin through 15 seconds, discard waste.
      g)Add 200 uL of Zymo Wash Buffer
      h)spin through 15 seconds, discard waste.
      i)spin for 90 seconds, full speed to dry.
      j)add 50 uL water and elute with water (spin 60s) into a fresh Eppendorf tube labeled " purified PCA1" [Note: the amount of water added depends on the starting amount of PCA product) 
  Now, the cleaned up assembly reaction as template for PCA2 is collected product into a fresh Eppendorf tube.
3. (Skip running on a gel due to smeary mess) Conduct amplification after zymo cleanup:
      a)label a PCR tube "PCA2 Lz-AAAA-1", then add:
               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
       b) Mix well and centrifuge briefly, then store on ice.
       c) save the purified PCA1 tube in the freezer in case this step fails!

***The next steps (programming) are running by GSI:
             a) 2 min initial denature at 94°C
             b) 30 sec denature at 94°C
             c) 30 sec anneal at 60°C [This should be high, as your outer oligos now have a huge overlap with the correct product]
             d) 30 sec extension at 68°C
             e) repeat 2-4 30 times total

Huiting He 15:05, 21 February 2012 (EST)

==Zymo Cleanup of Tar PCR product and Lz-AAAA PCA2 product==



===Zymo Cleanup of Tar PCR product===
Since the expected size of PCR Tar product is 802bp, choose regular Zymo Cleanup Procedure to 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 ~30uL Tar PCR product.
   2.Transfer into the Zymo column (small clear guys)
   3.spin through (15sec), discard waste.
   4.Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
   5.spin through(15sec), discard waste.
   6.Add 200 uL of Zymo Wash Buffer
   7.spin through(15sec), discard waste.
   8.spin for 90 seconds, full speed to dry.
   9.elute with water(30uL) into a fresh Eppendorf tube, (the same volume of water as the volume of the original reaction) store in ice.



===Analytical gel of PCA2 product OF Lz-AAAA===
1.Obtain my PCA2 product of Lz-AAAA
2.label tube "PCA2-AAAA", mix 6uL PCA2-Lz-AAAA product and 4uL dye, then load into the well(#1) of gel2 prepared by GSI. <br>
3.run the gel for about 40 mins, then take a picture.


===Zymo Cleanup of Lz-AAAA PCA2===
Since the size of PCA2 is 139bp<300bp, choose the  small-Frag Zymo Cleanup to removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction for fragments smaller than 300bp. It also will remove the buffer and restriction enzymes from a restriction digest reaction
   1.Add 100 uL of Zymo ADB buffer (brown bottle) to the PCA2 tube.
   2.Transfer into the Zymo column (small clear guys)
   3.Add 500uL of Ethanol and pipette up and down to mix
   4.spin through (15s), discard waste.
   5.Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
   6.spin through (15s), discard waste.
   7.Add 200 uL of Zymo Wash Buffer
   8.spin through, discard waste.
   9.spin for 90 seconds, full speed to dry.
   10.elute with water 8uL (Since we add 8uL at the beginning of digestion) spin for 60s into a fresh Eppendorf tube, and store on ice.

Huiting He 12:56, 23 February 2012 (EST)

==NheI/BamHI Digest PCR product Tar and NheI/BamHI wobble digest Lz-AAAA==

===NheI/BamHI Digest PCR product Tar, run the gel purification===
1. Digest PCR product Tar 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.
6. transfer into the Zymo column inside a collection tube (small clear guys)
7.spin through (15 sec), discard waste.
8. Add 200 uL of Zymo Wash Bbuffer (which is basically 70% ethanol)
9. spin through (15 sec), discard waste.
10. Add 200 uL of Zymo Wash Buffer
11. spin through (15 sec), discard waste.
12. spin for 90 seconds, full speed to dry.
13. elute with water 8uL and spin for 60 seconds into a fresh Eppendorf tube and store on ice [Tar digpdt]


===NheI/BamHI Digest of Wobble Products Lz-AAAA===
Since the size of Lz-AAAA is small fragment, use the wobble digest
1. set up the following reaction in a PCR tube:
      50uL eluted Lz-AAAA
      5.7uL NEB Buffer 2
      0.5uL NheI
      0.5uL BamHI
2. Mix thoroughly by slamming the tube upside down on the table, and then a quick spin to move the liquid to the bottom of the tube.
3. Incubate the reaction at 37 degrees on the thermocycler
4. Proceed to another Zymo small fragment cleanup
        a)     Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
	b)	Transfer into the Zymo column (small clear guys)
	c)	Add 500uL of Ethanol and pipette up and down to mix
	d)	spin through (15s), discard waste.
	e)	Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
	f)	spin through (15s), discard waste.
	g)	Add 200 uL of Zymo Wash Buffer
	h)	spin through, discard waste.
	i)	spin for 90 seconds, full speed to dry.
	j)	elute with water 50uL (spin 60s) into a fresh Eppendorf tube and store on ice [lz-AAAA-digpdt]

Huiting He 18:38, 24 February 2012 (EST)

==Ligation of NheI/BamHI digests and transformation of Tar and Lz-AAAA individually==
The procedure are same for  these two products(Tar and Lz-AAAA)

===Ligation of NheI/BamHI digests===
1.Set up the following reaction:
          6.5uL ddH2O
          1uL T4 DNA Ligase Buffer (purple striped tubes)
          1uL Vector digest (pBca9525-sbb1384 for both Tar and Lz-AAAA, done by GSI)
          1uL Insert digest (Tar-dig or Lz-dig)
          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 100 uL aliquot of cells on ice
  2. Add 25 uL of water to the cells
  3. Add 15 uL of KCM to the cells 
  4. Put my ligation mixtures(Tar-dig and Lz-dig) on ice, let cool a minute or two 
  5. Add 70 uL of the cell cocktail (aliquot of cells+25 uL of water+15 uL of KCM) 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
 10. label the plate(Tar,2/24/2012 and Lz-AAAA, 2/24/2012)
 11. plate 75 uL on selective antibiotics (flame the tips and plate cover, add 75 uL in plate evenly and wait for 3mins to dry) 
 12. incubate at 37 degrees UPSIDE DOWN overnight (GSI will freeze it after 24 hours)

Huiting He 12:56, 28 February 2012 (EST)

since the culture of all of 4 tubes of Lz-AAAA1 and 3 tubes of Tar are clear after colonies incubate overnight (the colonies were picked up by GSI), I need to redo the ligation and transformation. 



Ligation of NheI/BamHI digests and transformation of Tar and Lz-AAAA individually
The procedure are same for  these two products(Tar and Lz-AAAA)

Ligation of NheI/BamHI digests
1.Set up the following reaction:
          6.5uL ddH2O
          1uL T4 DNA Ligase Buffer (purple striped tubes)
          1uL Vector digest (pBca9525-sbb1384 for both Tar and Lz-AAAA, done by GSI)
          1uL Insert digest (Tar-dig or Lz-dig)
          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 100 uL aliquot of cells on ice
  2. Add 25 uL of water to the cells
  3. Add 15 uL of KCM to the cells 
  4. Put my ligation mixtures(Tar-dig and Lz-dig) on ice, let cool a minute or two 
  5. Add 70 uL of the cell cocktail (aliquot of cells+25 uL of water+15 uL of KCM) 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 40 minutes
 10. label the plate(Tar,2/24/2012 and Lz-AAAA, 2/24/2012)
 11. plate 75 uL on selective antibiotics (flame the tips and plate cover, add 75 uL in plate evenly and wait for 3mins to dry) 
 12. incubate at 37 degrees UPSIDE DOWN overnight (GSI will freeze it after 24 hours)


Miniprep purification of Tar 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.
Spin in centrifuge at top speed for 5 minutes.
Label blue columns with an alcohol-resistant lab pen.
Pour liquid into columns, and place the columns into the centrifuge. Spin at full speed for 15 seconds.
Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
Wash each column with 500 uL of PB buffer.
Spin in centrifuge at full speed for 15 seconds, then flick out the liquid again.
Wash with 750uL of PE buffer (washes the salts off the resins).
Spin in centrifuge at full speed for 15 seconds and flick out liquid again.
Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol.
Label new Microcentrifuge tubes and put columns in them.
Elute them by adding 50uL of water down the middle of the column (don't let it stick to the sides).
Spin in centrifuge at top speed for 30 seconds.
Take out columns and cap the tubes.





[User:Huiting He|Huiting He]] 12:56, 1 March 2012 (EST)





Huiting He 15:44, 2 March 2012 (EST)