IGEM:Hong Kong HKUST/Investigations/Robustness of colony PCR compared to regular PCR

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Contents

Authors

Aim

To determine if the use of colony instead of extracted plasmid DNA hinders generation of PCR products

Introduction

Polymerase Chain Reaction (PCR) is a routine biochemical technology used to amplify pieces of DNA for various applications, including preliminary screening, DNA cloning, sequencing and functional analysis of genes. Among different types of PCR, colony PCR allows rapid screening of bacterial colonies for correct DNA vector constructs. While primers have direct access to extracted plasmid in regular PCR, bacterial cells are lysed in the denaturation step of colony PCR process for primers to bind to ssDNA. Assuming that all conditions and reagents are constant, this investigation aims to determine if the use of colony instead of extracted plasmid affects generation of PCR products.
In this investigation, the same bacterial cell was inoculated for colony and regular PCR. After running PCR under the same condition1, the PCR product was analyzed by running on agarose gel electrophoresis. This investigation aims to determine validity in the use of colony PCR for prescreening and other potential applications.
1 For colony PCR, initial denaturation time increased from three to five minutes (suggested by NEB).

Description of Actions

Experiment Flow
Experiment Flow


Description of Actions
The validity of colony PCR was determined by comparing PCR products using colony with extracted plasmid (positive control) and ddH2O (negative control). A single colony with pSB1C3 - RFP coding device (BBa_J04450) was isolated and streaked onto agar LB plates to be used for colony and regular PCR. For regular PCR, the colony was inoculated to be grown in LB liquid broth for 16 hours in 37 degrees Celsius. Plasmids were then extracted from grown bacterials cells using GTpure extraction kit. NanoDrop was executed to determine the concentration of DNA for PCR mixture preparation. Using a colony, extracted plasmids and ddH2O, PCR was executed using VF2 and VR for forward and reverse primers respectively. PCR products were then analyzed by running agarose gel electrophoresis with 140V electricity for 30 minutes, along with 1kb ladder, template DNA (BBa_J04450 - pSB1C3) and template DNA digested with EcoRI and PstI, which served as references for the PCR products. After the gel was post-stained in ethidium bromide for 40 minutes, DNA bands of colony and regular PCR products were observed under ultraviolet light.


Results

Data: Agarose Gel Photos


Figure 1. Analyzing PCR products on agarose gels. The polymerase chain reaction (PCR) was run using the colony and extracted plasmid of pSB1C3 - RFP coding device (pSB1C3 - BBa_J04450) and ddH2O for negative control. The PCR products were then resolved on 1% agarose gel along with GeneRuler 1kb ladder, template plasmid (pSB1C3 - BBa_J04450) and template plasmid digested with EcoRI and PstI, and post-stained with ethidium bromide.  (1) Gel from Chloe. (2) Gel from Rinaldi. (3) Gel from Jesslyn.
Figure 1. Analyzing PCR products on agarose gels. The polymerase chain reaction (PCR) was run using the colony and extracted plasmid of pSB1C3 - RFP coding device (pSB1C3 - BBa_J04450) and ddH2O for negative control. The PCR products were then resolved on 1% agarose gel along with GeneRuler 1kb ladder, template plasmid (pSB1C3 - BBa_J04450) and template plasmid digested with EcoRI and PstI, and post-stained with ethidium bromide. (1) Gel from Chloe. (2) Gel from Rinaldi. (3) Gel from Jesslyn.

Using the same set of primers (VF2 and VR) for colony and regular PCR, the same size of RFP coding device was resolved on the gel. Taking reference from the DNA band of digested template DNA (1.1kb) and comparing with DNA bands resolved from ddH2O PCR products for negative control, bands of the colony and regular PCR products (1.4kb) show successful amplification of RFP coding device. For regular PCR products, remaining template plasmid was also resolved on the agarose gel, while colony PCR products had greater quantity of primers, which can be seen from Chloe’s and Rinaldi’s gel photos.


Possible Issues
  • On Jesslyn’s gel, the the template DNA (BBa_J04450) and product of regular PCR were lost in the process of DNA loading onto the gel. Additional volume (5uL) of regular PCR products were added into the column, thus Jesslyn's gel photo cannot be analyzed quantitatively
  • For Jesslyn’s PCR mixture using ddH2O, DNA was added accidently instead of ddH2O. Thus, the data cannot be trusted as negative control.


Conclusion

Based on DNA band analysis, colony and regular PCR successfully amplified the RFP coding device. This shows that the use of colony instead of extracted plasmid does not hinder generation of PCR products.


Protocol & References

  • Plasmid Extraction: using GTpure Plasmid Extraction Kit.
  • PCR Reaction Protocol
Media:PCR Reaction Protocol.pdf
  • Restriction Digestion:
Media:Restriction Digestion Protocol.pdf
  • Electrophoresis:
Media:Electrophoresis Protocol.pdf
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