Revision as of 16:45, 25 July 2011

This protocol was developed by Tom Knight, samples of standard Registry plasmid backbones prepared using this method were sent out in the Spring 2011 DNA Distribution kits.

Short single stranded DNA fragments will not ligate to 4 bp overhangs. By creating a very short overhang on a PCR of a plasmid backbone, the remnant, when cut with EcoRI and PstI is sufficiently short that it will not anneal at ligation temperature, and will therefore not ligate. This allows us to build high quality construction plasmid backbone without purifying away the cut fragments remaining after PCR.

We are distributing the prepared construction plasmid as purified PCR products, diluted to standard concentration, but prior to cutting with EcoRI and PstI. Standard assembly will cut this plasmid backbone with EcoRI and PstI at the same time that the two assembled fragments are cut with EcoRI and SpeI and with XbaI and PstI, respectively.

The preparation of this PCR fragment is done with primers having short overhangs past the EcoRI and PstI sites, followed by PCR cleanup, dilution to standard concentration, and quality control testing.

Note: The Registry shipping plasmid backbone is pSB1C3. If you are making linearized plasmid backbone in order to send parts to the Registry, you must use pBS1C3.

Primers:

gccgctgcagtccggcaaaaaa,SB-prep-3P-1
atgaattccagaaatcatccttagcg,SB-prep-2Ea

Diluted to 30 pmol/ul

These primers have been tested with pSB1C3, pSB1A3, pSB1K3, and pSB1T3.

Using the Linearized Plasmid Backbones

The Spring 2011 DNA Distribution should come with a set of linearized plasmid backbones: pSB1A3, pSB1C3, pSB1K3, and pSB1T3. The linearized plasmid backbones (25ng/ul at 50ul) should be stored at 4C or lower. Prior to ligation the plasmid backbones need to be cut with EcoRI and PstI.

Digest

• Enzyme Master Mix for Plasmid Backbone (25ul total, for 6 rxns)
  • 5 ul NEB Buffer 2
  • 0.5 ul BSA
  • 0.5 ul [http://www.neb.com/nebecomm/products/productR3101.asp EcoRI-HF]
  • 0.5 ul [http://www.neb.com/nebecomm/products/productR0140.asp PstI]
  • 0.5 ul [http://www.neb.com/nebecomm/products/productR0176.asp DpnI] (Used to digest any template DNA from production)
  • 18 ul dH20

• Digest Plasmid Backbone
  • Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)
  • Add 4 ul of Enzyme Master Mix
  • Digest 37C/30 min, heat kill 80C/20 min

Ligation

• Add 2ul of digested plasmid backbone (25 ng)
• Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 ul)
• Add equimolar amount of XbaI PstI digested fragment (< 3 ul)
• Add 1 ul [http://www.neb.com/nebecomm/products/productm0202.asp T4 DNA ligase buffer]. Note: Do not use quick ligase
• Add 0.5 ul [http://www.neb.com/nebecomm/products/productm0202.asp T4 DNA ligase]
• Add water to 10 ul
• Ligate 16C/30 min, heat kill 80C/20 min
• Transform with 1-2 ul of product

Making Linearized Plasmid Backbones

Bulk Production

The following is the protocol that we used to create the linearized plasmid backbones shipped with the Spring 2011 DNA Distribution. The protocol is in 96 well format, but may be scaled down to suit smaller batches.

2011 Plasmid Backbone Production

PCR mix

• 9.6ml of [http://products.invitrogen.com/ivgn/product/10790020?ICID=search-10790020 PCR Supermix High Fidelity]
• 67 ul of primer SB-prep-2Eb
• 67 ul of primer SB-prep-3P-1
• 10 ul of template DNA at 10ng/ul (100ng total)
  • Notes:
  1. Do not use a sample of linearized plasmid backbones (PCRed) as a template,
  2. The Registry uses plasmid backbones with a BBa_J04450 insert as a template
• Aliquot 100ul per well in 96 well plate

PCR program

1. 95C/2min
2. 95C/30s
3. 55C/30s
4. 68C/3min
5. Repeat cycle (steps 2 to 4, 37 more times)
6. 68C/10min

PCR cleanup

Purification of 96 well plates was done through Promega [http://www.promega.com/products/dna-and-rna-purification/dna-fragment-purification/wizard-sv-96-pcr-clean_up-system/ Wizard SV 96 PCR Clean-Up kit] and a vacuum manifold. The protocol below follows the manual, with a few changes (in bold), however please see manual for setup instructions.

1. Add equal volume of Binding Solution to PCR product (add 100ul of Binding Solution to 100ul of product)
2. Mix by pipetting, transfer all 200ul to Binding Plate, let sit for 1 min
3. Apply vacuum until samples pass through, about 30s to 1 min
4. Add 200 ul of freshly prepared 80% ethanol to Binding Plate, let sit for 1min, apply vacuum until ethanol passes through, about 20s to 1 min.
5. Repeat ethanol wash (step 4) twice more for three washes total
6. Remove Binding Plate from wash manifold, blot on kim wipes, reinstall in wash manifold
7. Apply vacuum for 4 min to fully dry Binding Plate
8. Remove Binding Plate from wash manifold, blot on kim wipes, reinstall in collection manifold
9. Add 50ul of TE buffer, let sit for 1 min, apply vacuum until eluted, about 1 min
10. Repeat 50ul elution (step 9) for a total elution of 100ul
11. Measure concentration on nanodrop, adjust to 25 ng/ul with TE

Single Reaction PCR

PCR mix

• 100 ul [http://products.invitrogen.com/ivgn/product/10790020?ICID=search-10790020 PCR Supermix High Fidelity]
• 0.7 ul of SB-prep-3P-1
• 0.7 ul of SB-prep-2Ea
• 0.5 ul template DNA at 10 ng/ul
  • Notes:
  1. Do not use a sample of linearized plasmid backbones (PCRed) as a template,
  2. The Registry uses BBa_J04450 as a template

PCR program

1. 94C/2min
2. 94C/30s
3. 55C/30s
4. 68C/3min
5. Repeat cycle (steps 2 to 4, 35 more times)
6. 68C/10min
7. Digest with DpnI enzyme: 2ul in 100ul reaction, incubate 37C/hour; heat kill 80C/20min

PCR cleanup

[http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickpcrpurificationkit.aspx QIAquick PCR Purification]

• Add 500 ul Qiagen buffer PB
• Spin through a column twice, discard flowthrough
• Wash 1x with 700 ul buffer PB
• Wash 2x with 760 ul buffer PE
• Discard liquid, spin dry at 17000g for 3 min
• Elute into a new tube twice with 50 ul of TE (100 ul total)

Quality Control

We recommend QCing constructed linearized plasmid backbones, to test success of PCR, ligation efficiency, and background.

1. Run unpurified PCR product (1 ul) on a gel to verify the correct band and concentration and lack of side products.
2. Test concentration of purified PCR product. Note: Expected yield should be 40ng/ul or higher. Adjust to 25ng/ul with TE.
3. Run a digest and ligation test with purified PCR product to determine EcoRI and PstI cutting and ligation efficiency.

Digest

• Digest Master Mix (10rxns)
  • 15 ul NEB Buffer 2
  • 1.5 ul BSA
  • 90 ul dH20

• Run Digest
  • 4 ul of plasmid backbone (approximately 100 ng)
  • 10.5 ul of Digest Master Mix
  • 0.5 ul either EcoRI-HF or PstI enzyme (not both!)
  • Digest 37C/30min; 80C/20 min
  • Proceed directly to ligation

Ligation

• Ligation Master Mix (10rxns)
  • 20 ul T4 DNA ligase buffer
  • 5 ul T4 DNA ligase
  • 25 ul water

• Ligation Test
  • Add 5 ul of ligation master mix to digested product
  • Ligate 16C/30min; 80C/20 min
  • Run all 20 ul on a gel
  • Compare intensity of the single and double length bands. More efficient ligations will show stronger double length bands than single.

Transformation test

• Transform 1 ul of the diluted final product into highly competent cells
• Control transform 10 pg of pUC19
• Plate on the appropriate antibiotic
• Observe few colonies. Any colonies represent background to the three antibiotic assembly process
• Quantify the effective amount of remaining circular DNA able to transform

Linearized Plasmid Backbones Original Protocol