Team:Stanford/Notebook/Lab Work/Week 8

Spring: Brainstorming | Spring Meetings

Summer: Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Summaries

8/16 Monday

Francisco's Notebook

Promoter - RSID, sRNA ligations Redesigned oligos (contains entire sequence, has restriction enzyme sticky ends)

Karina's Notebook

Need to redesign oligos! It seems that there is a sequence of 16 bp of the "rev-comp scar + scar" that is forming homodimers and selfdimers. 16 bp is significant since our annealing region for the primers is ~20 bp, thus a lot of unwanted byproduct is forming in the PCR stitching reaction. Instead of stitching our parts, we will design our oligos so that they are each the full length of the strand and anneal the two strands together. We will also design our oligos so that they anneal with the XbaI and PstI overhangs, enabling us to skip the restriction digest step.

To redesign!

8/17 Tuesday

Francisco's Notebook

Promoter - RSID, sRNA ligations

• Redesigned oligos (contains entire sequence, has restriction enzyme sticky ends)
• Inoculated pBad and pLux; two tubes each, 6 ml culture in each tube

8/18 Wednesday

Francisco's Notebook

Promoter - RSID, sRNA ligations

Redesigned oligos (contains entire sequence, has restriction enzyme sticky ends)

Miniprepped pBad and pLux; used Zippy kit, eluted in 30 uL Qiagen EB (10mM Tris-Cl)

Diagnostic Gel of pBad and pLux miniprep

• Gel Estimates (ng/uL):
  • pBad 1: 50, pBad 2: 40, pLux 1: 40, pLux 2: 100
• Nanodrop Results for comparison (ng/uL):
  • pBad 1: 89.1, pBad 2: 85.3, pLux 1: 32.0, pLux 2: 97.3

Digestion of pLux and pBad at E, S (for 3A assembly)

• Recipe:
  • Used all of pBad 1 and pLux 2 DNA, added water to top off 34 uL
  • (25 uL pBad 1 + 9 uL water, 26 uL pLux 2 + 8 uL water)
  • 5.0 uL BSA
  • 5.0 uL NEBuffer 4
  • 3.0 uL SpeI (added 3:45pm, heat inactivate 9:50am next day)
  • 3.5 uL EcoRI (added 10:15am next day)

Karina's Notebook

Our order for the redesigned oligos came back because of a series of poly-G runs found in the sRNA 2 Reverse and sRNA 2C Reverse. More than 4 G's in a row forms what is called a "guanine tetracomplex" that is highly unstable knot. The poly-G runs are editable and we'll have to edit our designs and resubmit.

8/19 Thursday

Promoter - RSID, sRNA ligations Diagnostic Gel of pLux and pBad

• promoters cut in many places - had 3 or 4 bands.

8/20 Friday

Francisco's Notebook

Promoter - GFP, RFP - terminator ligations

Was going to 3A assemble promoters, reporter and terminator - but there is not RBS (it is coming with the RSID) Anyways, still Digested GFP-term 1, RFP-term 1 at X, P (2 hour digestion)

• G1 ~113 ng/uL --> 25 uL
• R1 ~114 ng/uL --> 25 uL

Digested promoters at E, S (2 hour digestion)

• pBad 2 85 ng/uL --> 23 uL (only had this much left)
• pLux 1 32 ng/uL --> 23 uL (only had this much left)

Digested J64100 (ccdb, C resistance) at E, P

• 69 ng/uL --> 30 uL