Alberta NINT/5 June 2008


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lab work (JD):

             Purified gel band of K102001/PstI and SpeI from yesterday.  
             Collected quantitative data on K102001, E0433, and null output.  
             Performed Ligation of K102001+E0433=K102003 and K102001+null=K102005.  
              Transformed bacteria and plated them.

lab work (SD):

             Inoculated 6 LB + amp50 culture tubes with colonies from K102009 plate (04/06/08).
             Incubated at 37 C, shaking, overnight.

Plates K102008 and K102008 negative control had >100 colonies each. Re-try digest using NEB 2 buffer. Purified using MinElute PCR purification standard protocol. Ligated K102002 /B+N with T/A 0 /B+N.

lab work (WM):

            Made K102034, 2035 and 2036 to test which anti-sense strand is best activator of K102031.

2034 is complementary to 5' stem and 5' half of loop 2035 is complementary to all of loop and a few nt's both 5' and 3' of loop 2036 is complementary to 3' half of loop and 3' stem

These are to be inserted into K102011 in place of the current null input (TA0In). This is replaced by HindIII+NcoI digest in a manner similar to the standard BioBrick digest. We can still use 2 ul 10XNEB2 buffer in 2ul 10XBSA in a 20 ul reaction.

Single stranded oligo's were purchased from IDT then annealed and digest with HindIII+NcoI to make the inserts. Digests were purified with the Qiagen QiaQuick PCR Purification kit, resulting in 8,000 - 10,000 fmols/ul of digested inserts. These were diluted 1/100 and inserted into the digested K102011/HindIII+NcoI.

Then 1.5ul of the ligation was transformed into 100 ul XL1-B cells.