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Edinburgh/20 July 2008
From 2008.igem.org
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Week 5
Sunday 20 July 08
- Ran P24 and P25 on Gel 19. No amplification products. Time to do some trouble shooting. The fault is in either the primers, the template, or the reactions. All previous reactions have been fine, so most likely the primers or template. To check this, we need to do a positive control using universal primers such as fd1 and rd1 (amplify 16S rRNA gene rrnB) which should definitely give a product. If these fail, then it is most likely that the template is at fault - we should prepare genomic DNA and try using this as template rather than cells; also do a Gram stain to confirm that the cells look OK. If the control reaction works, try remaking the primer solutions, though it seems unlikely that all four sets could be wrong, unless I was having a really bad day. (CF)
- Redid digest of M48 (pSB1A2+rbs+dxs) with SacI/SpeI. Does not look good. Also repeated digests of clones M36 (pSB1A2+crtB) and M42 (pSB1A2+crtI) alternating single digests (EcoRI) and double digests (EcoRI/PstI) to check for inserts. Look OK. Submit for sequencing. (CF)
- Also: I have a plan for in vitro assembly to cut down on transformations and minipreps. Could have a try of this next week if there are some students with some free time. (CF)
