Cloning DNA with pGEMT-Easy intermediate vector

Kok Zhi Lee
Aug 22 · 3 min read

— a rapid method to ligate your DNA without restriction enzymes

Purpose:

This method is used to ligate a piece of DNA to a commercialized vector, pGEMT-Easy when you have a limited amount of DNA. I used it as an intermediate cloning vector because this vector can produce a bunch of plasmids harboring my inserted DNA with its high-copy number nature.

Materials:

  1. NEB Taq polymerase: it contains 10x standard Taq buffer and Taq polymerase
  2. dNTP
  3. pGEMT-Easy kit: it contains pGEMT-Easy linearized vector, 2X Rapid Ligation Buffer, T4 DNA ligase, and control Insert DNA (you don’t need this if you are confident with your ligation)
  4. NEB T4 DNA ligase kit: it contains 10x ligation buffer and T4 ligase (I prefer using this because of 10x, so I have sufficient volume to put more insert, especially when the insert concentration is low.)

Procedures:

Adding A at the 3' end of your DNA insert.

  1. Resuspend a minimum amount of 100ng of blunt-end DNA (could be G-block or PCR product) with 44ul water in a PCR tube.
  2. Add 5 ul of 10x standard Taq buffer
  3. Add 1 ul of dNTP.
  4. Add 0.2 ul of Taq polymerase
  5. Incubate the PCR tube in a thermocycler at 72C for 30 minutes.
  6. Do PCR clean up and resuspend with 13 ul d2H2O.
  7. Use 1 ul to check the concentration.

Ligation

8. Prepare the following ligation reaction in a PCR tube:

DNA Insert with A’: 100–300 ng (I always use 200ng)

pGEMT-Easy: 0.5 ul

NEB 10x ligation buffer: 1 ul

NEB ligase: 1 ul

d2H2O: make the total volume* up to 10ul

*I sometimes make it up to 15 or 20ul; everything stays the same except 10x buffer needs to adjust.

9. Incubate the ligation product at 16C for at least 30 minutes (I always use 30 minutes) and it is ready to transform!

Blue-white screening

The beauty of this pGEMT-Easy vector is that the multiple cloning site (MCS) is within part of the lacZ gene, which encodes β-galactosidase. Successful DNA ligation in the MCS would disrupt the reading frame of lacZ, inactivating β-galactosidase. When plating with X-gal and IPTG, active β-galactosidase would catalyze X-gal into a blue product while inactive β-galactosidase failed to do so. So you can select the white colonies for subsequent screening.

Tips:

  1. X-gal: dissolved in DMSO; working concentration: 20ug/ml; should store in an amber tube (light-sensitive)
  2. IPTG: working concentration: 0.1mM
  3. Sometimes, we spread X-gal and IPTG on plate unevenly, resulting in some regions have darker blue colonies while some regions are pale blue. To avoid picking the wrong colonies, I always select white colonies around a blue colony. Presence of blue colonies indicates that the region has sufficient X-gal and IPTG, so the white colony is an actual white colony, not because of lacking X-gal and IPTG.
  4. Sometimes, you will get some pale blue colonies, even around blue colonies. This could have resulted from your short DNA fragment insertion within the MCS. I once have a fluorescent protein without a stop codon as an insert, and my colonies are pale blue.
  5. The direction of your ligated insert matters if your DNA encodes a toxic product. Remember they are two different configurations that can be inserted in the MCS. Given that lacZ is transcribed in one direction, your DNA would be mutated if your toxic gene is in frame with lacZ. If that is the case, screen more colonies, find the opposite configuration.
0.5 ul of pGEMT-Easy was used to ligate with my mysterious insert (200ng), resulting in >95% of successful white colonies.

Kok Zhi Lee

Written by

As a synthetic biologist, I study interesting biomolecules, pull them out of the native context, and repurpose them for different biotechnological applications.

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