IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week5/Chemical and Light

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(Analytical Digest of Composite Parts 7/22)
(Recombination Update 07/22)
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*Thus far, none of the attempts to extract flanking regions from the genomic DNA have worked.  We are troubleshooting this now and will run positive controls with known DNA samples to check the efficiency of our Phusion Polimerase.
*Thus far, none of the attempts to extract flanking regions from the genomic DNA have worked.  We are troubleshooting this now and will run positive controls with known DNA samples to check the efficiency of our Phusion Polimerase.
 +
 +
=07/23 Ligations/Transformations=
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*Ligated p40 cut SP with mtrB cut XP at 2:3, 1:2, 1:4, 1:5, 1:6, positive control is uncut p40, negative control (to test Amp plates) is p29 (Kan)

Revision as of 10:38, 23 July 2008

Contents

Testing Thermoinducible Lac System

Restreaked Plates of P92-3 in S1 7/21

Restreaked P93 (1:3), P92a, and P93b--all successfully transformed in S1--to detect YFP at 30 and 40 degrees.

Results 7/22

S1 P93 (1:3), P92a, and P93b grown at 30 and 40 degrees failed to demonstrate any difference in YFP expression. Both expressed very low, if any, YFP.

Sequencing Parts, Plasmids, Etc.

Primers for Sequencing Remy's Plasmids and Duet Vectors 7/21

Primer Name Sequence
P4P13fwdGGATCTCGACGCTCTCCCTT
P12fwdATGCGTCCGGCGTAGAGGA
DuetRevTGCTAGTTATTGCTCAGCGG

Analytical Digest of Composite Parts 7/22

Digested composite plasmids to verify size.

Plasmid DNA Added (uL) 10x Buffer 3 (uL) 100x BSA (uL) XbaI (uL) PstI (uL) Water (uL) Total (uL) < 1 ug DNA? Expected Size
S1 P75a 1 (7/22)72.5 uL0.25 uL1 uL1 uL13.2525 uL925bp, 2750bp
S1 P75a 2 (7/22)52.5 uL0.25 uL1 uL1 uL15.2525 uL925bp, 2750bp
S1 P75b 1 (7/22)72.5 uL0.25 uL1 uL1 uL13.2525 uL925bp, 2750bp
S1 P75b 2 (7/22)42.5 uL0.25 uL1 uL1 uL16.2525 uL925bp, 2750bp
E1 P92a 1:1 (7/17)72.5 uL0.25 uL1 uL1 uL13.2525 uL2314bp, 2750bp
E1 P92b 1:1 (7/17)72.5 uL0.25 uL1 uL1 uL13.2525 uL2314bp, 2750bp
E1 P93a 1:1 (7/17)72.5 uL0.25 uL1 uL1 uL13.2525 uL2314bp, 2750bp
E1 P93b 1:1 (7/17)102.5 uL0.25 uL1 uL1 uL10.2525 uL2314bp, 2750bp
E1 P92a PCR (7/18)152.5 uL0.25 uL1 uL1 uL5.2525 uLYes2314bp, 2750bp
S1 P93 1:3 1 (7/17)72.5 uL0.25 uL1 uL1 uL13.2525 uL2314bp, 2750bp
S1 P93 1:3 2 (7/17)72.5 uL0.25 uL1 uL1 uL13.2525 uL2314bp, 2750bp
S1 P92a PCR 1 (7/18)72.5 uL0.25 uL1 uL1 uL13.2525 uL2314bp, 2750bp
S1 P92a PCR 2 (7/18)72.5 uL0.25 uL1 uL1 uL13.2525 uL2314bp, 2750bp
S1 P93b PCR 1 (7/18)42.5 uL0.25 uL1 uL1 uL16.2525 uL2314bp, 2750bp
S1 P93b PCR 2 (7/18)72.5 uL0.25 uL1 uL1 uL13.2525 uL2314bp, 2750bp
E1 P58b 1 (7/18)72.5 uL0.25 uL1 uL1 uL13.2525 uL937bp, 2750bp
E1 P58b 2 (7/18)102.5 uL0.25 uL1 uL1 uL10.2525 uL937bp, 2750bp
S1 P58b A (7/15)102.5 uL0.25 uL1 uL1 uL10.2525 uLYes937bp, 2750bp
S1 P58b B (7/15)102.5 uL0.25 uL1 uL1 uL10.2525 uLYes937bp, 2750bp
S1 P59 (7/9)102.5 uL0.25 uL1 uL1 uL10.2525 uLYes1122bp, 2750bp
S1 P59b 1 (7/22)102.5 uL0.25 uL1 uL1 uL10.2525 uLYes1122bp, 2750bp
S1 P59b 2 (7/18)32.5 uL0.25 uL1 uL1 uL17.2525 uL1122bp, 2750bp

Making Thermoinducible cI Lambda System

Primers for Taking Out cI857 from PGW7 7/21

Primer Name Sequence
CI857_RBSfwdAtcgagaattcgcggccgcttctagagaaagaggagaaaatgagcacaaaaaagaaac
CI857_RBSrevAtcgatactagtagcggccgctgcagtcagccaaacgtctcttcag
CI857_BIGfwdAtcgagaattcgcggccgcttctagagtcatgacattaacctataaa
CI857_BIGrevatcgatactagtagcggccgctgcagcagccagcagagaattaagg

Minipreps of Transformed Parts in S1 7/22

Also includes transformed P28 and the thermoinducible lac system.

Plasmid Name ng/uL A260 260/280 260/230 Constant
S1 P75a 1156.23.1241.992.2750
S1 P75a 2203.244.0652.042.2350
S1 P75b 1177.413.5482.022.1650
S1 P75b 2258.855.1772.012.350
S1 P28a 1155.423.1082.032.2750
S1 P28a 2239.514.792.062.2650
S1 P28a 3204.54.092.012.2450
S1 P93 1:3 1175.273.5052.072.3250
S1 P93 1:3 2166.93.3382.122.3850
S1 P92a PCR 1152.583.0522.022.2850
S1 P92a PCR 2157.593.1522.022.2250
S1 P93b PCR 1291.875.8372.132.2350
S1 P93b PCR 21523.042.072.2150

Adding Terminator Before pLambda GFP

Digestion of Terminator (P64) and pLambda GFP 7/22

' P75a 1 (vector) P75b 1 (vector) P64a 07 (insert)
DNA10 uL10 uL10 uL
100x BSA0.25 uL0.25 uL0.25 uL
10x Buffer2.5 uL EcoRI buffer2.5 uL EcoRI buffer2.5 uL EcoRI buffer
Enzyme 11 uL EcoRI1 uL EcoRI1 uL EcoRI
Enzyme 21 uL XbaI1 uL XbaI1 uL SpeI
Water5.25 uL 5.25 uL 5.25 uL
Total25 uL25 uL25 uL

Housekeeping

Making Competent Cells 7/22

Made ~600 100uL (some were 200 uL, indicated by green dot on top) aliquots from 1L of culture. Done as per Jason's lab's protocol.

PCR

mtrB

7/22: gradient + new R primer

Rx mix (split into 12 samples): 180μL PCR supermix, 4μL mtrB-ApaLI-F primer (20μM), 4μL mtrB-KpnI-R-new (20μM), pipet tip touch of mtrB BB from gel purification, 12μL water Rx: 5min @ 94°C → 35x[45s @ 94°C → 45s @ {52-58 gradient}°C → 2m38s @72°C] → 5min @ 72°C → ∞ @ 4°C

No bands on gel from any of the conditions.

Rx mix (split into 12 samples): 180μL PCR supermix, 4μL mtrB-ApaLI-F primer (20μM), 4μL mtrB-KpnI-R-new (20μM), pipet tip touch of WT S. oneidensis MR-1, 12μL water Rx: 10min @ 94°C → 40x[45s @ 94°C → 45s @ {44-51 gradient}°C → 2m45s @72°C] → 5min @ 72°C → ∞ @ 4°C

RE digests 07/22

Gel 1

Image:7-22_gel_1_MXHTA.jpg

Lane 1: 1 kb plus ladder

Lane 2: CDF PCR cut XP (~900)

Lane 3: CDF PCR uncut (~900)

Lane 4: P1A cut XP (2750)

Lane 5: P1A uncut (3669)

Lane 6: P17 cut EX (5327)

Lane 7: P17 uncut (5327)

Lane 8: P48 cut XP (769)

Lane 9: P48 uncut (3477)


Gel 2

Image:7-22_gel_2_MXHTA.jpg

Lane 1: 1 kb plus ladder

Lane 2: P49B cut XP (943)

Lane 3: P49B uncut (3651)

Lane 4: P51 cut EX (5795)

Lane 5: P51 uncut (5795)

Lane 6: P52 cut EX (5412)

Lane 7: P52 uncut (5412)

Lane 8: P63 cut EX (3284)

Lane 9: P63 uncut (3284)


Gel 3

Image:7-22_gel_3_MXHTA.jpg

Lane 1: 1 kb plus ladder

Lane 2: P97A cut EX (2091)

Lane 3: P97A uncut (2091)

Lane 4: P97B cut EX (2091)

Lane 5: P97B uncut (2091)

Ligations 07/22

  • We ligated P1A (vector) to CDF. We used three different ratios of vector to insert for the ligation: 2/6, 1/5, 1/7.
  • Attempted to ligate p40 cut with SP (w/ RBS) to mtrB cut with XP. Thus far, the ligation has not worked.

Recombination Update 07/22

  • Thus far, none of the attempts to extract flanking regions from the genomic DNA have worked. We are troubleshooting this now and will run positive controls with known DNA samples to check the efficiency of our Phusion Polimerase.

07/23 Ligations/Transformations

  • Ligated p40 cut SP with mtrB cut XP at 2:3, 1:2, 1:4, 1:5, 1:6, positive control is uncut p40, negative control (to test Amp plates) is p29 (Kan)
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