Jamesh008:consensus DNA ligation protocol

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==General Information==
==General Information==
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This is a [[Jamesh008:consensus protocol|consensus protocol]] click to see what this means.
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This is a [[Jamesh008:consensus protocol|consensus protocol]].
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This protocol describes blunt end cloning into linearized plasmid vectors and subsequent transformation. [[Wikipedia:DNA ligase|DNA ligase]] is used to create a [[Wikipedia:phosphodiester bond|phosphodiester bond]] between the 5' phosphate and 3' hydroxyl groups of DNA.  Most commonly, one needs to insert a DNA sequence of interest into a plasmid, ready for [[transformation]] into [[competent cells]].  Ideally, DNA and vector are individually cut with the same restriction enzyme, then both are added to a ligation reaction to be circularised by DNA ligase.  [[T4 DNA ligase]] is the most commonly used [[Wikipedia:DNA ligase|DNA ligase]] for molecular biology techniques.<br>
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The two components of the DNA in the ligation reaction should be equimolar and around 100ug/ml. If the plasmid vector to target DNA ratio is too high then excess 'empty' mono and polymeric plasmids will be generated. If too low then the result may be an excess of linear and circular homo- and heteropolymers.<br>
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Following ligation you will transform the circularsed plasmid, now containing your insert DNA, into competent bacteria for further selection and analysis. Most bacteria do not usually exist in a “transformation ready” state, but the bacteria can be made permeable to the plasmid DNA, and cells that are capable of transformation are referred to as “competent.” Competent cells are extremely fragile and should be handled gently, specifically kept cold and not vortexed. The transformation procedure is efficient enough for most lab purposes, with efficiencies as high as 109 transformed cells per microgram of DNA, but it is important to realize that even with high efficiency cells only 1 DNA molecule in about 10,000 is successfully transformed.<br>
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During “transformation,” a single plasmid from the ligation mixture enters a single bacterium and, once inside, replicates and expresses the genes it encodes. One of the genes on the pCX-NNX plasmid leads to ampicillin-resistance. Thus, a transformed bacterium will grow on agar medium containing ampicillin. Untransformed cells will die before they can form a colony on the agar surface.
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[[Wikipedia:DNA ligase|DNA ligase]] is used to create a [[Wikipedia:phosphodiester bond|phosphodiester bond]] between the 5' phosphate and 3' hydroxyl groups of DNA.  Most commonly, one needs to insert a DNA sequence of interest into a plasmid.  Ideally, you can cut the DNA and vector with the same restriction enzyme.  Then you can add both DNA sequences, the cut insert and vector sequence, and ligate them together to form a circular vector using DNA ligase.  [[T4 DNA ligase]] is the most commonly used [[Wikipedia:DNA ligase|DNA ligase]] for molecular biology techniques.
 
==Materials==
==Materials==

Revision as of 12:12, 30 October 2006

Contents

General Information

This is a consensus protocol.

This protocol describes blunt end cloning into linearized plasmid vectors and subsequent transformation. DNA ligase is used to create a phosphodiester bond between the 5' phosphate and 3' hydroxyl groups of DNA. Most commonly, one needs to insert a DNA sequence of interest into a plasmid, ready for transformation into competent cells. Ideally, DNA and vector are individually cut with the same restriction enzyme, then both are added to a ligation reaction to be circularised by DNA ligase. T4 DNA ligase is the most commonly used DNA ligase for molecular biology techniques.
The two components of the DNA in the ligation reaction should be equimolar and around 100ug/ml. If the plasmid vector to target DNA ratio is too high then excess 'empty' mono and polymeric plasmids will be generated. If too low then the result may be an excess of linear and circular homo- and heteropolymers.
Following ligation you will transform the circularsed plasmid, now containing your insert DNA, into competent bacteria for further selection and analysis. Most bacteria do not usually exist in a “transformation ready” state, but the bacteria can be made permeable to the plasmid DNA, and cells that are capable of transformation are referred to as “competent.” Competent cells are extremely fragile and should be handled gently, specifically kept cold and not vortexed. The transformation procedure is efficient enough for most lab purposes, with efficiencies as high as 109 transformed cells per microgram of DNA, but it is important to realize that even with high efficiency cells only 1 DNA molecule in about 10,000 is successfully transformed.
During “transformation,” a single plasmid from the ligation mixture enters a single bacterium and, once inside, replicates and expresses the genes it encodes. One of the genes on the pCX-NNX plasmid leads to ampicillin-resistance. Thus, a transformed bacterium will grow on agar medium containing ampicillin. Untransformed cells will die before they can form a colony on the agar surface.


Materials

  • T4 DNA Ligase
  • 10x T4 DNA Ligase Buffer
  • Deionized, sterile H2O
  • Purified, linearized vector (likely in H2O or EB)
  • Purified, linearized insert (likely in H2O or EB)


Protocol Procedure

10μl Ligation Mix

Larger ligation mixes are also commonly used

  • 1.0 μL 10X T4 ligase buffer
  • 6:1 Molar ratio of insert to vector (~10ng vector)
  • Add (8.5 - vector and insert volume)μl ddH2O
  • 0.5 μL T4 Ligase

Calculating Insert Amount

 {Insert\ Mass\ in\ ng} = 6\times\left[\frac{{Insert\ Length\ in\ bp}}{{Vector\ Length\ in\ bp}}\right]\times{Vector\ Mass\ in\ ng}

This differs from the Knight calculation, not sure why, but it may be important.

Method

  1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube
  2. Add 1 μL ligation buffer to the tube.
    Vortex buffer before pipetting to ensure that it is well-mixed.
    Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.
  3. Add appropriate amount of insert to the tube.
  4. Add appropriate amount of vector to the tube.
  5. Add 0.5 μL ligase.
    Vortex ligase before pipetting to ensure that it is well-mixed.
    Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting.
  6. Let the 10 μL solution sit at 22.5°C for 30 mins
  7. Denature the ligase at 65°C for 10min
  8. Dialyze for 20 minutes if electroporating
  9. Use disks shiny side up
  10. Store at -20°C

Notes

  1. Make sure the buffer is completely melted and dissolved. Precipitate is DTT (or BSA?). Probably best to aliquot this buffer into smaller portions, to reduce the freeze/thaw cycles. In general, make sure the buffer still smells strongly like "wet dog" (Checking if the DTT is still good.)
  2. If you are having trouble with your ligation, NEB offers FAQ's (Quick Ligation T4 DNA ligase) to help.
  3. Prior to the ligation, some heat their DNA slightly (maybe ~37°C) to melt any sticky ends which may have annealed improperly at low temperatures.
  4. Tom Knight has read that ligase can inhibit transformation. By heat-inactivating the ligase, this inhibition can be avoided. However, according to the NEB FAQ, heat-inactivation of PEG (which is present in the ligation reaction) also inhibits transformation, therefore a spin-column purification is recommended prior to transformation if you are having problems.
  5. Treating PCR products with proteinase K prior to restriction digest dramatically improves the efficiency of subsequent ligation reactions. [1]

Please add discussion points with the prefix *''~~~~''

Please sign your name to your note by adding ('''~~~~''') to the end of your tip.

References

  1. Crowe JS, Cooper HJ, Smith MA, Sims MJ, Parker D, and Gewert D. . pmid:2011503. PubMed HubMed [Crowe-NAR-1991]

Specific Protocols

Endy:DNA ligation using T4 DNA ligase -- Using T4 DNA Ligase

Knight:DNA ligation using NEB Quick Ligation Kit -- 5min ligation.

Knight:TOPO TA cloning -- For PCR products.

Silver:Ligation -- A protocol using the Roche Kit.

BE.109:DNA ligation -- A ligation protocol for classroom use in a laboratory class taught at MIT. Uses T4 DNA ligase but has interesting tips and tricks.

Contact

  • Jamesh008 is the Jamesh008:consensus DNA ligation protocol editor.
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