SBB10Ntbk-WeiChunHsu

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Part Synthesis (aka Wet Lab)

Wei-Chun Hu 18:18, 24 February 2010 (EST)

Protocol changes

  • Change for protocol (replace with small fragment zymo cleanup) for PCA
  • Did not get to Digest step (advised to hold off due to running time, and given possible failure of 4ABC...)

Products

  • 4D - zymo-ed PCA (band looks fine, but faint)
  • 5D - incomplete digest reaction - contains 8 uL of 4D
  • 5ABC - zymo-ed SOEing PCR (problem: see below)
  • 6ABC - incomplete digest reaction - contains 8 uL of 5ABC

Potential problems

  • Band for 4ABC analytical gel is WAY off (5th from the left band is a bit over 500, product should be around 1800). Jeni also had the same problem with SOEing (band on far right). Recommended change SOEing protocol (SLIC, Venter?)

Wei-Chun Hu 15:31, 23 February 2010 (EST)

Protocol: PCA

  1. Optional: Run an analytical gel - 3 uL PCR, 7 uL loading buffer
    1. This gel is probably not necessary because we will do a gel in the digest step
    2. Band should be around 250 bp (below the 1st bright one)
  2. Clean up the amplification reactions with a zymo column
    1. Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
    2. Transfer into the Zymo column (small clear guys)
    3. Add 500uL of Ethanol and pipette up and down to mix
    4. spin through, discard waste.
    5. Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
    6. spin through, discard waste.
    7. Add 200 uL of PE or Zymo Wash buffer
    8. spin through, discard waste.
    9. spin for 90 seconds, full speed to dry.
    10. elute with water into a fresh Eppendorf tube
  3. Digest the product and desired backbone with Eco/Bam:
    1. Set up the following reaction (two tubes):
      1. 8uL of eluted PCR product (backbone [pBjk2741-Bca1144] and PCA product in SEPARATE reactions)
      2. 1uL of NEB Buffer 2
      3. 0.5uL EcoRI
      4. 0.5uL BamHI
    2. Incubate at 37 degrees on the thermocycler for 1hr
    3. 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
    4. Only proceed below if ample time
    5. Set up a Zymo column
      1. The DNA is shorter than 300bp: add 250uL of isopropanol and mix prior to loading it on the column

Protocol: SOE

  1. Optional: Run an analytical gel - 3 uL PCR, 7 uL loading buffer
    1. Expect bright band around 1800 bp (just above the middle bright one)
  2. Clean up the amplification reactions with a zymo column
    1. Add 180 uL of Zymo ADB buffer (brown bottle) to the reaction.
    2. Transfer into the Zymo column (small clear guys)
    3. spin through, 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
  3. Digest the product and desired backbone with Eco/Bam:
    1. Set up the following reaction (we already have the plasmid digest):
      1. 8uL of eluted PCR product (PCR product)
      2. 1uL of NEB Buffer 2
      3. 0.5uL EcoRI
      4. 0.5uL BamHI
    2. Incubate at 37 degrees on the thermocycler for 1hr
    3. 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

Wei-Chun Hu 17:31, 22 February 2010 (EST)

Results

Gel for PCR


Last three columns are A,B,C. Lengths appear to be correct (~900,~500,~450). Distortion of gel makes it impossible to accurately compare the last two.

Protocol changes

  • 8uL6uL of PCR product premixed with 1uL4uL of loading buffer
  • Used Jeni's mastermix for 4ABC PCR because frankly everyone was running out of time

Products

  • PCA
    • 2D - zymo cleanup of 1D
    • 3D - PCR amplification of 2D (put in PCA box)
  • SOE
    • JHA,JHB,JHC - preparative gels of 1A,1B,1C (6 uL PCR product / 4 uL 1X loading buffer)
    • 2ABC - purifying gel fragments 1A,1B,1C
    • 3ABC - purified 50uL DNA from gel fragments
    • 4ABC - PCR reaction using 0.5 uL of 3ABC (put in 2K55 box)

Wei-Chun Hu 18:21, 21 February 2010 (EST)

Protocol: SOE

(This assumes the PCR has completed successfully.)

  • For each PCR, load 6uL of PCR product premixed with 4uL of loading buffer in a single well of a 1% agarose gel
  • Cut out the bands, put them into a single 1.5mL microcentrifuge tube (put ALL bands in one tube)
  • Add 650uL of ADB Buffer

Zymo gel purification

  • All spins until the drying step are 15 second full speed spins.
  1. cut out bands minimizing extra gel matter.
  2. put in ependorf tube and add 600uL of Zymo ADB buffer (brown bottle).
  3. heat at 55, shake and/or vortex until the gel has dissolved.
  4. (do NOT put isopropanol.)
  5. transfer into the Zymo column inside a collection tube (small clear guys)
  6. spin through, discard waste.
  7. Add 200 uL of PE buffer (which is basically 70% ethanol)
  8. spin through, discard waste.
  9. Add 200 uL of PE buffer
  10. spin through, discard waste.
  11. spin for 90 seconds, full speed to dry.
  12. discard collection tube
  • Elute the DNA in 50uL of water in a LABELED eppendorf tube
  • Dilute 10 uM stocks of OJIMH011 and OJIMH014.
  • Set up your second round of PCR as a normal 33uL reaction using the eluted mixture of fragments as template:
24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL OJIMH011, 10uM (dilute 100 uM stock)
1uL OJIMH014, 10uM (dilute 100 uM stock)
0.5uL Expand polymerase "1"
0.5uL Template DNA

Protocol: PCA

Small-Frag Zymo Cleanup

The following procedure 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 reaction.
  2. Transfer into the Zymo column (small clear guys)
  3. Add 500uL of Ethanol and pipette up and down to mix
  4. spin through, discard waste.
  5. Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
  6. spin through, discard waste.
  7. Add 200 uL of PE or Zymo Wash buffer
  8. spin through, discard waste.
  9. spin for 90 seconds, full speed to dry.
  10. elute with water into a fresh Eppendorf tube

Amplification

Now, you need to do an amplification of the correct full-length chunks. Clean up the assembly reaction with a zymo column; don't bother running it on a gel - it'll be a smeary mess and won't really help you. Save the purified product in case this step fails! For the amplification reaction, do a normal phusion program with 1 ul of the cleaned up assembly reaction as template, and using the outermost oligos for the chunk. That is:
Recipe

  1. 1 ul each outer oligo [sbb11 F/R] (10 uM)
  2. .5 ul phusion
  3. 10 ul 5x phusion buffer
  4. 5 ul 2mM dNTPs
  5. 32.5 ul H2O

Program

  1. 2 min initial denature at 94oC
  2. 30 sec denature at 94oC
  3. 30 sec anneal at 60oC [This should be high, as your outer oligos now have a huge overlap with the correct product]
  4. 30 sec extension at 68oC
  5. repeat 2-4 30 times total

Wei-Chun Hu 17:33, 17 February 2010 (EST)

Protocol

Resuspend lypophilized DNA. Oligo stock is 100 uM, dilute to 10 uM.
Appropriate components (see Media:2-17-10-cloning-PCR.pdf) were added to three Eppendorf tubes. Transfer to PCR tubes at end. DNA polymerase was added to the mastermix.

Oligos for PCA were provided as mixture (100 uM, M), forward (10 uM, F), and reverse (10 uM, R). The Template:SBB-PCA protocol was used.

Products

1A: OJIMH011,OJIMH013,Bca1623 6-9
1B: OJIMH012,CA1674,Bca1559 7-7
1C: CA1675,OJIMH014,Bca1559 7-1
1D: Oligo mixture (M SBB11)

Wei-Chun Hu 18:10, 16 February 2010 (EST)

Run this protocol tomorrow: Media:2-17-10-cloning-PCR.pdf

Wei-Chun Hu 14:24, 9 February 2010 (EST)

The following protocols are relevant for me:

Cloning by PCR
SOEing PCR
PCA Gene Synthesis

Regular Zymo Cleanup
Zymo Gel Purification

EcoRI/BamHI Digest of PCR Products
Ligation of EcoRI/BamHI digests

Transformation by heat-shock
Picking of colonies
Miniprep purification of DNA

Computational Part Design

Wei-Chun Hu 01:45, 4 February 2010 (EST)

All construction files (now transcluded)

1) sbb11: sleeping beauty 5'TR

Pool OJIMH001 through OJIMH010, assemble by PCA
PCR OJIMH001/OJIMH010 on PCA reaction	(257 bp, EcoRI/BamHI)
Sub in pBjk2741-Bca1144	(EcoRI/BamHI, 910+2170, L)
Product is pBjk2741-sbb11

------------

OJIMH001	PCA assembly of sbb11	CCATAGAATTCATGAGATCTAGTTGAAGTCGGAAGTTTACATACACTTAAG
OJIMH002	PCA assembly of sbb11	GAAAAACGAGTTTTAATGACTCCAACTTAAGTGTATGTAAACTTCCGACTTC
OJIMH003	PCA assembly of sbb11	AAGTTGGAGTCATTAAAACTCGTTTTTCAACTACACCACAAATTTCTTGTTAAC
OJIMH004	PCA assembly of sbb11	CTGACTTGCCAAAACTATTGTTTGTTAACAAGAAATTTGTGGTGTAGTTGAAA
OJIMH005	PCA assembly of sbb11	TTAACAAACAATAGTTTTGGCAAGTCAGTTAGGACATCTACTTTGTGC
OJIMH006	PCA assembly of sbb11	ACAATTGTTGGAAAAATGACTTGTGTCATGCACAAAGTAGATGTCCTAACTG
OJIMH007	PCA assembly of sbb11	GACACAAGTCATTTTTCCAACAATTGTTTACAGACAGATTATTTCACTTATAATTC
OJIMH008	PCA assembly of sbb11	CCACTGGAATTGTGATACAGTGAATTATAAGTGAAATAATCTGTCTGTAAAC
OJIMH009	PCA assembly of sbb11	TAATTCACTGTATCACAATTCCAGTGGGTCAGAAGTTTACATACACTAAG
OJIMH010	PCA assembly of sbb11	CTGATGGATCCTTAGTGTATGTAAACTTCTGACCC

JCA Notes

  • Correct
2) sbb13: PhiC31 Integrase

PCR OJIMH011/OJIMH012 on pBca9523-Bca1623	(949 bp, gp = A)
PCR OJIMH013/OJIMH014 on pBca9523-Bca1659	(938 bp, gp = B)
-------------
PCR PJIMH011/OJIMH014 on A+B	(1843 bp, EcoRI/BamHI)
Sub in pBjk2741-Bca1144	(EcoRI/BamHI, 910+2170, L)
Product is pBjk2741-sbb13	{<phiC31>}
-------------
OJIMH011	Forward PCR of Part 1 of PhiC31	ccataGAATTCatgAGATCTGACACGTACGCGGGTGCTTA
OJIMH012	SOEing of Part 1 (R) of PhiC31	AAGAAGCCGGACGGCACGCCGACCacgaagattgagggttaccg
OJIMH013	SOEing of Part 2 (F) of PhiC31	cggtaaccctcaatcttcgtGGTCGGCGTGCCGTCCGGCTTCTT
OJIMH014	Reverse PCR of Part 2 of PhiC31	atcagGGATCCCGCCGCTACGTCTTCCGT

JCA Notes

  • Oligos 12 and 13 are switched
  • Otherwise correct
sbb13: PhiC31 Integrase

PCR OJIMH011/OJIMH013 on Bca1623 6-9            (949bp, gp=A)
PCR OJIMH012/CA1674 on Bca1559 7-7              (514bp, gp=B)
PCR CA1675/OJIMH014 on Bca1559 7-1	        (444bp, gp = C)
-------------
PCR PJIMH011/OJIMH014 on A+B+C	(1843 bp, EcoRI/BamHI)
Sub in pBjk2741-Bca1144	(EcoRI/BamHI, 910+2170, L)
Product is pBjk2741-sbb13	{<phiC31>}
-------------
OJIMH011	Forward PCR of Part 1 of PhiC31	
ccataGAATTCatgAGATCTGACACGTACGCGGGTGCTTA
OJIMH012	SOEing of Part 1 (R) of PhiC31	
AAGAAGCCGGACGGCACGCCGACCacgaagattgagggttaccg
OJIMH013	SOEing of Part 2 (F) of PhiC31	
cggtaaccctcaatcttcgtGGTCGGCGTGCCGTCCGGCTTCTT
CA1674	GGGCGTCGGCGCGCTCCGCAACAAGGTTCGCCCGTTCGCCGCTCTTCTCAG	
PCA assembly of phiCthreeprime (Bca1659)
CA1675	TGCGGAGCGCGCCGACGCCCTGAACGCCCTTGAAGAGCTGTACGAAGACCG	
PCA assembly of phiCthreeprime (Bca1659)
OJIMH014	Reverse PCR of Part 2 of PhiC31	
atcagGGATCCCGCCGCTACGTCTTCCGT


Wei-Chun Hu 20:45, 3 February 2010 (EST)

Construction file for sbb13 (this one is especially tricky, so please check)

2) sbb13: PhiC31 Integrase

PCR OJIMH011/OJIMH012 on pBca9523-Bca1623	(949 bp, gp = A)
PCR OJIMH013/OJIMH014 on pBca9523-Bca1659	(938 bp, gp = B)
-------------
PCR PJIMH011/OJIM014 on A+B	(1843 bp, EcoRI/BamHI)
Sub in pBjk2741-Bca1144	(EcoRI/BamHI, 910+2170, L)
Product is pBjk2741-sbb13	{<phiC31>}
-------------
OJIMH011	Forward PCR of Part 1 of PhiC31	ccataGAATTCatgAGATCTGACACGTACGCGGGTGCTTA
OJIMH012	SOEing of Part 1 (R) of PhiC31	AAGAAGCCGGACGGCACGCCGACCacgaagattgagggttaccg
OJIMH013	SOEing of Part 2 (F) of PhiC31	cggtaaccctcaatcttcgtGGTCGGCGTGCCGTCCGGCTTCTT
OJIMH014	Reverse PCR of Part 2 of PhiC31	atcagGGATCCCGCCGCTACGTCTTCCGT

Wei-Chun Hu 20:03, 3 February 2010 (EST)

Construction file for sbb11 (check for errors)

1) sbb11: sleeping beauty 5'TR

Pool OJIMH001 through OJIMH010, assemble by PCA
PCR OJIMH001/OJIMH010 on PCA reaction	(257 bp, EcoRI/BamHI)
Sub in pBjk2741-Bca1144	(EcoRI/BamHI, 910+2170, L)
Product is pBjk2741-sbb11

------------

OJIMH001	PCA assembly of sbb11	CCATAGAATTCATGAGATCTAGTTGAAGTCGGAAGTTTACATACACTTAAG
OJIMH002	PCA assembly of sbb11	GAAAAACGAGTTTTAATGACTCCAACTTAAGTGTATGTAAACTTCCGACTTC
OJIMH003	PCA assembly of sbb11	AAGTTGGAGTCATTAAAACTCGTTTTTCAACTACACCACAAATTTCTTGTTAAC
OJIMH004	PCA assembly of sbb11	CTGACTTGCCAAAACTATTGTTTGTTAACAAGAAATTTGTGGTGTAGTTGAAA
OJIMH005	PCA assembly of sbb11	TTAACAAACAATAGTTTTGGCAAGTCAGTTAGGACATCTACTTTGTGC
OJIMH006	PCA assembly of sbb11	ACAATTGTTGGAAAAATGACTTGTGTCATGCACAAAGTAGATGTCCTAACTG
OJIMH007	PCA assembly of sbb11	GACACAAGTCATTTTTCCAACAATTGTTTACAGACAGATTATTTCACTTATAATTC
OJIMH008	PCA assembly of sbb11	CCACTGGAATTGTGATACAGTGAATTATAAGTGAAATAATCTGTCTGTAAAC
OJIMH009	PCA assembly of sbb11	TAATTCACTGTATCACAATTCCAGTGGGTCAGAAGTTTACATACACTAAG
OJIMH010	PCA assembly of sbb11	CTGATGGATCCTTAGTGTATGTAAACTTCTGACCC

Wei-Chun Hu 19:45, 3 February 2010 (EST)

A test entry.

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