IGEM:Hong Kong HKUST/Investigations/Effects of ATP concentration to the Ligation Efficiency/Entry Base: Difference between revisions
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<br><br><b>Table 1- digestion recipe of plasmid J04450-pSB3K3.</b> S1, S2, S3, S4 are referred as experimental setups, while S1-C, S2-C, S3-C, S4-C are controls.<br> | <br><br><b>Table 1- digestion recipe of plasmid J04450-pSB3K3.</b> S1, S2, S3, S4 are referred as experimental setups, while S1-C, S2-C, S3-C, S4-C are controls.<br> | ||
"http://openwetware.org/images/e/ec/Table_1_from_R%26S.PNG" | "http://openwetware.org/images/e/ec/Table_1_from_R%26S.PNG" | ||
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<b>Table 2- digestion recipe of plasmid B0015-pSB1AK3</b> | <b>Table 2- digestion recipe of plasmid B0015-pSB1AK3</b> |
Revision as of 21:07, 7 June 2015
AuthorsAbstractLigase is an important enzyme involved in joining nicks of DNA strands, especially in forming recombinant DNA. As ATP is required as a co-factor of T4 DNA ligase activity, an experiment was done investigating the effect of ATP concentration on DNA ligation success rate. Two DNA fragments obtained from the BioBrick Parts Registry -BBa_J04450, an mRFP generator, and pSB1AK3, which is a vector with high copy number- were ligated in ligation mixtures with different ATP concentrations.
IntroductionLigation is a process in which two fragments of DNA are joined together in the presence of enzyme ligase. T4 DNA ligase is one of the the enzymes commonly used for ligation in laboratories. It requires ATP as a co-factor to form phosphodiester bonds between the two DNA fragments. In the assembly of genes, optimisation of the ligation process is crucial for obtaining maximal successful DNA constructs. Thus, in this experiment, the effect of ATP concentration on ligation success rate was investigated.
Methods and Material1. Preparation of LB broth
2 bottles of 400mL of LB broth with agar were prepared, each by mixing the below ingredients and made up to 400ml with double distilled water(ddH2O).
- Tryptone 4g
- Yeast 2g
- NaCl 4g
- Agar 6g
The mixtures were brought to autoclave. One bottle was added with chloramphenicol and the other with ampicillin, then poured into plates and left for solidification.
Table 2- digestion recipe of plasmid B0015-pSB1AK3
"http://openwetware.org/images/7/74/Table_2_from_R%26S.PNG"
Table 3- ligation recipe
"http://openwetware.org/images/e/e2/Table_3_from_R%26S.PNG"
9. Transformation
The ligation mixtures after overnight incubation were each transformed into 50 µl of competent cells. The cell mixture was left to incubate for 1 hour at 37 °C to allow sufficient time for recovery.
ResultsThe following results were obtained after transforming the ligated products and incubating the transformed cells overnight.
ConclusionThrough data obtained in this experiment, a conclusion for the investigation topic could not be made. This is due to the insufficient sample replicates, which was because of the lack of pSB1AK3 obtained after the purification of gel following gel electrophoresis. In future experiments, more DNA should be used in digestion taking into account that loss of DNA occurs during electrophoresis. A discrepancy of data was observed for 15mM ATP ligation mixture. A significant decrease was observed compared to the 10mM ATP ligation mixture, from 642 to 6 CFU. This result could possibly be due to the following errors: 1) When the 100ul cells were split into half, proper mixing was missed, leading to a non-homogenous cell mixture. The cell solution that was transformed by 15mM ligation mixture had much lower cell number, leading to the low CFU on the agar plate. 2) Although all the transformations were done by the same person, there might be some discrepancies between these transformations, some mistakes or errors affected the 15mM transformation. |