IGEM:Hong Kong HKUST/Investigations/ Identifying Candidate Colonies by Colony PCR
Identifying Candidate Colonies by Colony PCR
To identify candidate colonies with the desired insert and backbone with colony PCR instead of regular PCR.
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. In the denaturation step of colony PCR process, bacterial cells are lysed for primers to bind to ssDNA. Then gel electrophoresis was used to identify the suitable PCR product.
In this investigation, an insert and a backbone were digested from separate plasmids and then ligated together. It was then allowed to transform overnight, and colonies were picked for colony PCR. Then the PCR products were analyzed by running on agarose gel electrophoresis. This investigation aims to determine successful ligation of the respective insert and backbone.
Methods and Materials
For this experiment, pSB1C3-BBa_C0012 plasmid and pSB3K3-BBa_I0500 plasmid were selected to digest and ligate into pSB3K3-BBa_C0012. Suitable candidates were further identified by colony PCR.
For digestion, one positive experimental setup and one negative control were prepared for each plasmid sample, namely, pSB1C3-BBa_C0012 plasmid and pSB3K3-BBa_I0500 plasmid. For the first set of positive and negative setups, they both contained 300 ng of the pSB1C3-BBa_C0012 plasmid, while the second set of positive and negative setups contained 1000 ng of the pSB3K3-BBa_I0500 plasmid. XbaI and SpeI-HF were used in digestion and were not added to the negative control. Instead, it was compensated by an additional 0.4 μl of MQ to keep the total digestion volume at 18 μl in accordance with the protocol. Both sets of positive setup and negative control were incubated at 37°C. After 2 hours of digestion, gel electrophoresis was carried out to inspect the digestion result.
A band size of 1153 bp was identified for the digested BBa_C0012 insert, while a band size of 2750bp was identified for the digested pSB3K3 backbone. The DNA bands were then cut and extracted by gel purification. The concentrations of the purified DNA were checked with Nanodrop, giving concentrations of 10.2 ng/μl for BBa_C0012 insert and 11.6 ng/μl for pSB3K3 backbone.
A set of positive experimental setup and a negative control were prepared for the ligation of the retrieved BBa_C0012 insert and pSB3K3 backbone. Both the positive and negative setups contained 71.4 ng of the BBa_C0012 insert and 57.188 ng of the pSB3K3 backbone. T4 ligase was used in ligation and was not added to the negative control. Instead, it was compensated by an additional 0.75 μl of MQ to keep the total ligation volume at 15 μl. Both the positive setup and the negative control were incubated at room temperature for an hour.
All 15 μl of the positive setup and 15 μl of the negative control were transformed into E. coli separately and spread on agar plates were allowed to grow overnight in a 37°C incubator.
Colony PCR was carried out to inspect the ligation results. Candidate colonies were identified by comparing PCR products using colonies from the positive setup’s agar plate with extracted plasmid (positive control) and colony from the negative control’s agar plate. 10 colonies were picked from the positive setup’s agar plate as candidates while 1 colony was picked from the negative control’s agar plate for negative control in colony PCR. Colony PCR was executed using VF2 and VR for forward and reverse primers respectively. PCR products were then analysed by running gel electrophoresis with 130V electricity for 30 minutes, along with 1kb plus ladder. DNA bands of colony PCR products were then observed under ultraviolet light.