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Team:Stanford/Notebook/Lab Work/Week 8
From 2010.igem.org
(→Francisco's Notebook) |
(→8/18 Wednesday) |
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== 8/18 Wednesday == | == 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=== | ===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. | 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. | ||
Revision as of 03:38, 25 October 2010
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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.
