Team:Yale/Our Project/Notebook/Week 7

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lab notebook: week 7 (7/19-7/25)

• Monday 7/19--Investigation of how pre-ligation processing affects ligation results
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Gel Purification of PCR product

• Ran the phsABC PCR product on a 1.0% agarose gel at 90 V versus a 1 kb ladder to see how clean the result is--see very strong bands at 3.6 kb as expected. Excised the bright band and ran a standard vacuum manifold gel extraction protocol to give three samples with DNA concentrations of 73.1, 43.3, and 45.8 ng/uL
Self-ligation of phsABC to test restriction enzymes

• Took the single enzyme digests from 7/16 and ran a self-ligation of each with 17 uL of DNA solution, 2 uL of ligase buffer, and 1 uL T4 ligase. In each case ran trials both with iGEM ligase and buffer and with Grindley lab ligase & buffer. Let the reactions run for 20 minutes at room temperature before heat-killing with 20 minutes at 80˚C
Dual-purpose gel

• Ran a gel to accomplish two things: 1.Determine whether the phsABC fragment was still present after gel extraction 2. Determine based on dimer formation of singly digested phsABC fragments whether the digestion/ligation is working well. In a 1.0% agarose gel run at 90 V had the following lane assignments, left to right: Lane 1--1 kb ladder, Lane 2--circular 8 kb pSB74 plasmid, Lane 3--1 kb ladder, Lane 4--phsABC from gel extraction, Lane 5--self ligation of SpeI-digested phsABC (iGEM ligase), Lane 6--self ligation of SpeI-digested phsABC (Grindley lab ligase), Lane 7--self ligation of EcoRI-digested phsABC (iGEM ligase), Lane 8--self ligation of EcoRI-digested phsABC (Grindley lab ligase)
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Self-ligation of singly digested phsABC fragments
• Each enzyme (AvaII, SpeI, and EcoRI) was used to digest both phsABC that had been purified by gel extraction and phsABC that had been purified by PCR clean-up. Now each of the six resulting solutions is in turn used in a self-ligation reaction containing 17 uL of the digested DNA, 2 uL of T4 ligase buffer, and 1 uL T4 ligase. These ligations are run for four hours at 16˚C to see if a longer ligation time at a lower temperature will yield better results.
Serial digestion of ligation components
• Will try running long sequential digests of both the phsABC insert and the B0015 vector. Will also try omitting CIP. The first B0015 digestion contains of 5 uL of NEB buffer 4, 0.5 uL BSA, 9.7 uL B0015 (at 103.3 ng/uL, total of 1 ug), 1.8 uL XbaI, and 33 uL of water. The first phsABC digestion has 5 uL of NEB buffer 4, 0.5 uL BSA, 7.6 uL of phsABC (1 ug DNA total, derived from PCR purification protocol), 1.8 uL of SpeI, and 35.1 uL of water. Both digestions were run for 4 hours at 37˚C before a twenty minute heat-kill at 80˚C.
• Rather than going through a long purification protocol between steps, simply added 0.5 uL of 5 M NaCl to each digestion so that the salt content fit the parameters needed by EcoRI, then added 1.8 uL of EcoRI and let run another four hours at 37˚C before heatkilling as before.
This day's labwork is also recorded on pages 75 & 76 of the hard copy lab notebook
Wednesday 7/21--analysis of self-ligation, tranformation of Biobrick Q04121, and set up of ligation attempt #6
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Q04121

• Having settled on Q04121 as an all-in-one promoter system of choice, transform the Biobrick into homemade Xl1-Blue competent cells according to the standard transformation procedure and plate on Kanamycin plates.
Gel of phsABC self-ligation efforts
• Ran a 1.0% agarose gel with the six self-ligation reactions. They were run at 90 V against a 1 kb ladder and pSB74.
From left to right the lanes are: Lane 1--1 kb ladder, Lane 2--circular pSB74 (about 8 kb), Lane 3--ligation of SpeI digest of gel extracted phsABC, Lane 4--Lane 3--ligation of SpeI digest of PCR clean-up phsABC, Lane 5--ligation of AvaII digest of gel extracted phsABC, Lane 6--ligation of AvaII digest of PCR cleanup phsABC, Lane 7--ligation of EcoRI digest of gel extracted phsABC, Lane 8--ligation of EcoRI digest of PCR clean-up phsABC

• The AvaII bands are too faint to see really, but the SpeI and EcoRI ligations appear to show bands near 7.3, where dimers would show if they formed, suggesting that the longer ligation time may have helped. In general though, the gel never resolved well even when the loading dye was run off the end.

Ligation using serial digestion products

This day's labwork is also recorded on pages 76-78 of the hard copy lab notebook
Thursday 7/22--
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Extra content This day's labwork is also recorded on page 79 of the hard copy lab notebook
Friday 7/23--
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Extra content This day's labwork is also recorded on pages 79 & 80 of the hard copy lab notebook
Saturday 7/24--
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Extra content This day's labwork is also recorded on page 81 of the hard copy lab notebook
Sunday 7/25--
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Extra content This day's labwork is also recorded on page 81 of the hard copy lab notebook