IGEM:MIT/2006/Notebook/2006-6-21: Difference between revisions
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==SAMT Sequencing Results== | ==SAMT Sequencing Results== | ||
#All of them failed- there must be issue with SAMT reverse primer | #All of them failed- there must be issue with SAMT reverse primer | ||
==SA Optimal Concentration== | |||
#No carbon source for the plates so no cell growth | |||
==To Do== | ==To Do== | ||
#Make new LB plates at optimal concentration of SA (with IPTG in media) | #Make new LB plates at optimal concentration of SA (with IPTG in media) | ||
#ligate the cut BSMT into a biobrick backbone | #ligate the cut BSMT into a biobrick backbone | ||
Revision as of 07:55, 21 June 2006
SAMT Sequencing Results
- All of them failed- there must be issue with SAMT reverse primer
SA Optimal Concentration
- No carbon source for the plates so no cell growth
To Do
- Make new LB plates at optimal concentration of SA (with IPTG in media)
- ligate the cut BSMT into a biobrick backbone
- use red Kan backbone
- ligation reaction temperature is a compromise between competing reactions -- room temperature is pretty effective and fast (15 minutes for transformants)
- note: T4 is optimal at 37c, but cannot run reaction at this temp b/c will get melting at this high of a temp. (sticky ends only have 8 H-bonds)
- note: could alternatively try 18c/16c in fridge overnight
- be kind to T4 buffer, can be less kind to T4 enzyme
- want equal moles of insert and backbone (50 nanograms each)
- run in smallest volume possible
- run ATF1 pcr products on gel to see if pcr was successful at higher temperature
- repeat BAMT and SAMT PCR at a higher temperature gradient
- try 48-64 this time and place most tubes in middle region
