|Cloning of mreB from Caulobacter crescentus into E. coli|| Main project page|
Previous entry Next entry
Exp. 5 Digestion of Mini Prep and Exp. 6 Preparatory Digest & Purification
This week we finished up experiment 5 by performing a digestion of the mini preps. By digesting our mini preps with EcoR1 and Pst1, we were able to separate our plasmid backbone from the promoter sequence. This gel picture shows our results:
M = DNA 100bp Plus Ladder, 1 = A1, 2 = A2, 3 = A3, 4 = A4, 5 = K1, 6 = K2, 7 = K3, 8 = K4, 9 = positive control 1, 10 = positive control 2. Samples A1-A4 are part BBa_K20600, samples K1-K4 are part BBa_I0500, and our positive controls were from PSB1A7. Part BBa_K20600 gave us a bright band at about 2000bp, which is the plasmid backbone. Another very faint band was found around 150bp - this is the promoter sequence. There was some undigested DNA around 1500bp. Part BBa_I0500 showed faint bands above 3000bp, which is the plasmid backbone. There was also a slimmer, hardly detectable band around 1000bp, which is the promoter sequence for this part. Lastly, the control - PSB1A7, showed 2 distinct bands at 2000bp and 1000bp. We believe that the band around 1000bp is undigested, circular DNA because there is no promoter sequence. All of these bands came out at the expected length, but we are unsure why they are not as intense as the positive controls.
Today (Thursday), we digested and purified part BBa_K20600 with Spe1 and Pst1 in order to prepare our part for ligation with our gene. We then purified the part using the GeneJET PCR Purification Kit and obtained two, 35μL samples. The first sample will be more concentrated than the second sample. Our future plans include running a gel to check the digestion and compare the intensity/quantity of our part and gene using Mass Ruler marker.