IGEM:Imperial/2010/AmyE Vector

Starting from the top, we are assembling the first two fragments (K14070 and K14064) and K147002 with our oligos to add in a Dif site. Two dif sites on either side of resistance cassettes can be used to later excise antibiotic resistance from our final constructs.



- DNA was taken out of the reigstry - Cut with restriction enzymes - Run on a gel to confirm correct cutting and estimate relative ratios of DNA for ligation - Ligated overnight - Transformed into E.Coli to Amplify the DNA - Colony PCR has been used to confirm the correct insert size.
 * K14070 and K14064 fragments


 * Next Steps:

- Extract the DNA with a miniprep - Proceed to the next step - Reverse PCR



- Ligated two single stranded oligos together to produce Dif and insertion site - Standard biobrick assembly of oligos to K14002 - Ligation and transformation into E.coli competent cells (strain) - Screen plated colonies for correct insertion
 * K14002 and oligos

- Purify the correct insert out of E.coli - Next step assembly - LacI test vector and Final assembly vector
 * Next Steps



- Currently waiting for Part B step 2 Midi-prep results to start this step
 * LacI Testing vector