Difference between revisions of "Team:Edinburgh/Notebook/PMADetection"

PMA Detection

Week 1
Take Peroxidase out of the registry.
Order MAOA in two parts, N and C terminals.


Week 3
16/06
Digest pSB1C3 (1.23O2), pSB1A3 (4.2H1) and pSB1K3 (4.6B1) with EcoRI HF and PstI. Digest MAOA N and MAOA C (EcoRI HF/PstI).
Gel purify backbones, PCR purify inserts.


17/06
Ligate MAOA N 1 to pSB1C3, MAOA N 2 to pSB1K3, and MAOA C to pSB1A3. Transform.


18/06
Growth appears on all plates (except for negative control). Less growth on the plates with backbone only which indicates that some of the insert ligated into the vector.
Culture 3 colonies from each plate.


19/06
Miniprep cultures.
Diagnostic digest to show if inserts are actually present. pSB1K3 seems to have false positives.


Week 4
22/06
Prepare miniprep (MAOA N1 and MAOA C) for sequencing.


Week 5
29/06
Digest MAOA N1 1 in pSB1C3 and MAOA C 1 in pSB1A3 for fusion. PCR purify pSB1C3+MAOA N, gel purify MAOA C.
Ligate pSB1C3+MAOA N to MAOA C overnight.


30/06
Transform ligation reactions.


01/07
All of the transformations appeared to have worked. Culture transformations


02/07
Miniprep cultures.
Send pSB1C3+ MAOA N+MAOA C 1 and 2 for sequencing.
Run a diagnostic digest on them.
Diagnostic digest shows that MAOA N and C did not fuse, as together they should be around 2.3 kb and the insert is much smaller, closer to 1 kb which is the size of MAOA N alone.
Culture 4 more colonies from pSB1C3+MAOA N+MAOA C to see if there was successful fusion in any of them.


03/07
Miniprep the cultures.
Run diagnostic digest to see if the fusion worked.
Diagnostic gel shows that the fusion did not work.


Week 6
07/07
Retry fusing MAOA together. Digest pSB1C3 +MAOA N (AgeI/SpeI) and pSB1A3+MAOA C (NgoMIV/SpeI). Treat pSB1C3+ MAOA N with Antarctic phostphatase.
Gel purify.
Ligate MAOA C into pSB1C3+MAOA N.


08/07
Transform ligation results to see if MAOA fused.


09/07
No growth on plates which indicates that MAOA did not fuse.
Try fusion again by going back to the gBlock. Digest pSB1C3+MAOA N (AgeI/SpeI) and the MAOA C gBlock (NgoMIV/SpeI). Treat pSB1C3+MAOA N with Antarctic Phosphatase. Ligate the two together. Transform the ligation.


10/07
No colonies on control plates (backbone only), quite a few colonies on plates of backbone plus insert which indicates fusion of MAOA.


12/07
Inoculate colonies of transformants.


Week 7
13/07
Miniprep the two colonies with potential fusion of MAOA.
Diagnostic digest the plasmids to fully ensure fusion of MAOA.