IGEM:Hong Kong HKUST/Investigations/Performance of NotI-HF in 5, 15 and 30 minutes
For normal practice, researchers digest DNA using restriction enzyme for 1 to 1.5 hours. However, it is found that in NEB website, it claims that there are some enzymes which are qualified for TimeSaver™ can digest DNA for only five minutes, thus we would want to investigate on the shortest time that restriction enzyme can digest DNA so to reduce time cost for future researches. We choose to use linear DNA for this investigation, which is BBa_J04450.
Figure 1. Our experiment flowchart
Figure 2. BBa_J04450 plasmid with XbaI restriction enzyme site from iGEM Registry. http://beta.labgeni.us/registries/parts_registry/?part=BBa_J04450
Figure 3. NotI-HF restriction enzyme site in pSB1C3 from NEBcutter V2.0
To find out whether 200ng of DNA can be digested by using 0.2 μL of restriction enzyme in 5, 15 and 30 minutes.
Since DNA ladder is smearing, it is not helpful to identify the size of DNA samples.
From the negative control, the linear BBa_J04450, it shows two bands, which may be caused by the presence of other DNAs, but not solely BBa_J04450. The open circular BBa_J04450 has a smaller electrophoretic mobility, so it moves slower, and migrates less distance.
For our experimental DNA samples, most of them showed four bands, in which the first band all have the same number of base pair, which are higher than 3000 base pair, showing that this may be an unexpected DNA fragment, as the number of base pair of BBa_J04450 is 3139 bp. Therefore, the first band should be ignored when considering BBa_J04450.
For all our DNA samples, they have at least two bands for DNA, whereas the two bands showing that the DNA is digested. For some samples, three bands are obtained from the DNA after digestion, the three bands are at about 1500-3000 bp location, it showed that the DNA are not completely digested. While for those that only have two bands, they are almost completely digested, as cutting of linear BBa_J04450 produces two fragments of DNA. However, we cannot be sure that the DNA digested is BBa_J04450, as the summation of the base pair of the two bands are not equal to the number of base pair we should have for the original linear BBa_J04450.
Problems and limitations
1. Miserable results obtained which cannot be explained (gel photo)
2. Not enough apparatus so one have to wait for others to finish, which hinders the progress
3. Not familiar with the lab technique which can affect the final result
4. Did not prepare enough DNA for the digestion investigation
5. No linear DNA at the beginning and limited time to do experiments
6. The condition of our DNA digestion process might not exactly the same as the one NEB required
7. Gel purification kit which result in low concentration of DNA after purification
DNA is digested by enzyme in 5, 15 and 30 minutes, but all of the DNA cannot be completely digested by within the period of time so it is not recommended to digest DNA for only 5,15, and 30 minutes.
1. Transform competent cell (DH10B) with mRFP1 (BBa_J04450)
2. Prepare liquid LB medium
3. Cultivate E.Coli DH10B strain carrying BBa_J04450 in LB medium
4. Extract plasmid using GTpure™ Miniprep Kit
5. Quantify DNA concentration using NanoDrop
6. Digest BBa_J04450 using XbaI to get linear DNA
7. Run gel electrophoresis for the digested DNA to check whether the DNA is fully digested or not
8. Digest pSB1C3 using NotI-HF for 5, 15 and 30 minutes
9. Run gel electrophoresis to obtain the investigation results, and to examine the completeness of digestion