Team:Yale/Our Project/Notebook/Week 5

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lab notebook: week 5 (7/5 -7/11)

• Monday 7/5--Colony PCR of Ligation Attempt #2 Transformants
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Colony PCR

• Ran colony PCR of colonies 1B-9B from ligation attempt #2 according to the same protocol as used on 7/1.
• Then ran a 1.0% agarose gel of the nine PCR reaction solutions against a 1 kb ladder, but the resulting gel failed to show any evidence of either insert or vector, so will have to rely on sequencing and digests of miniprepped plasmids.
Gel of colony PCR from ligation attempt #2 Lane 1: 1 kb ladder, Lanes 3-11:PCR products from colonies 1B-9B, in order

• Miniprepped liquid cultures grown from colonies of first ligation attempt (colonies 1A-4A) and second ligation attempt (colonies 1B-9B) according to standard miniprep protocol.

This day's activities are also recorded on page 47 and 48 of the hard copy lab notebook.
• Tuesday 7/6--Analysis of Results of Ligation Attempts 1 & 2
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More Diagnostics of Ligation Results

• In order to determine success of ligation attempts 1 and 2, digested the miniprepped plasmids 1A-4A and 1B-9B from 7/5 with XbaI and SpeI and then looked for evidence of two fragments, one at 3.2 kb and the other at 3.6 kb. Reactions were conducted for 2 hours at 37°C, with the following contents for each of the thirteen reactions

Component	Volume
Distilled Water	6.4 μL
10x NEB Buffer 4	2 μL
DNA solution	10 μL
NEB XbaI	0.8 μL
NEB SpeI	0.8 μL
Total 20 μL

• The diagnostic 1.0% agarose gel was run at 90 V vs. a 1 kb ladder. Each of the thirteen samples gave one diffuse band around 3 kb and no evidence of the 3.6 kb phsABC fragment, so both ligation attempts #1 and #2 were unsuccessful.
Gel of XbaI and SpeI double digest of plasmids from ligation attempts #1 and #2 Lane 1: 1 kb ladder, Lanes 2-5:1A-4A, Lanes 6-14: 1B-9B

• During digestion prepped plasmid samples for sequencing. Each sample contained 3 μL of plasmid soln (average concentration of 96 ng/μL), 2 μL of 4 mM VF2 (forward primer), and 13 μL of distilled water. Intended to follow up with reverse sequencing of more promising samples, but gel results made it a moot point.

Wetlab work for this day is also recorded on pages 47, 49, and 50 in the hard copy lab notebook.
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