provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. Digest pSB1C3 using NotI-HF for 5, 15 and 30 minutesĩ.Takara Bio USA, Inc. Run gel electrophoresis for the digested DNA to check whether the DNA is fully digested or notĨ. Digest BBa_J04450 using XbaI to get linear DNAħ. Quantify DNA concentration using NanoDropĦ. Extract plasmid using GTpure™ Miniprep Kitĥ. Cultivate E.Coli DH10B strain carrying BBa_J04450 in LB mediumĤ. Transform competent cell (DH10B) with mRFP1 (BBa_J04450)ģ. Gel purification kit which result in low concentration of DNA after purificationĭNA 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.ġ. The condition of our DNA digestion process might not exactly the same as the one NEB requiredħ. No linear DNA at the beginning and limited time to do experimentsĦ.
Did not prepare enough DNA for the digestion investigationĥ. Not familiar with the lab technique which can affect the final resultĤ. Not enough apparatus so one have to wait for others to finish, which hinders the progressģ. Miserable results obtained which cannot be explained (gel photo)Ģ. 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.ġ. While for those that only have two bands, they are almost completely digested, as cutting of linear BBa_J04450 produces two fragments of DNA. 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.
Therefore, the first band should be ignored when considering BBa_J04450.įor all our DNA samples, they have at least two bands for DNA, whereas the two bands showing that the DNA is digested. The open circular BBa_J04450 has a smaller electrophoretic mobility, so it moves slower, and migrates less distance.įor 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. Since DNA ladder is smearing, it is not helpful to identify the size of DNA samples.įrom 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. To find out whether 200ng of DNA can be digested by using 0.2 μL of restriction enzyme in 5, 15 and 30 minutes. NotI-HF restriction enzyme site in pSB1C3 from NEBcutter V2.0 BBa_J04450 plasmid with XbaI restriction enzyme site from iGEM Registry. We choose to use linear DNA for this investigation, which is BBa_J04450.įigure 2. 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. For normal practice, researchers digest DNA using restriction enzyme for 1 to 1.5 hours.
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