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

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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 plates grown at 30 and 40 degrees failed to demonstrate any difference in YFP expression. Both expressed very low, if any, YFP.

Thermoinducible Test 7/24

' ' ' Time ' ' ' ' ' ' ' ' ' ' ' '
0 hrs2 hrs4 hrs6 hrs
StrainReplicateBefore Splitting3030 + 1mM IPTG40423030 + 1mM IPTG40423030 + 1mM IPTG4042* DID NOT SHAKE
LBN/ADilutionN/AN/AN/AN/AN/AN/AN/AN/AN/AN/AN/AN/AN/A
OD0000000000000
YFP36.7840.7240.7240.7240.7241.3241.3241.3241.3245.445.445.445.4
P84ADilutionN/A1 in 41 in 41 in 4.51 in 4.51 in 51 in 51 in 5.51 in 5.51 in 61 in 5.51 in 6.51 in 5.5
OD0.2690.2290.2180.2160.2140.2410.2240.2270.2190.2430.2340.2430.224
YFP291.95242.89226.53216.85211.21226.95222.07196.83196.3250.22238.06224.98215.35
YFP/OD1085.3159851060.6550221039.128441003.935185986.9626168941.7012448991.3839286867.092511896.3470321029.7119341017.350427925.8436214961.3839286
BDilutionN/A1 in 41 in 41 in 4.51 in 4.51 in 51 in 51 in 5.51 in 5.51 in 61 in 5.51 in 6.51 in 5.5
OD0.2690.2210.2180.2030.2080.2310.2280.2210.2280.2330.230.2280.268
YFP291.95225.35229.19208.3199.34215.49208.55195.39192.62233.83233.99215.07241.39
YFP/OD1085.3159851019.6832581051.3302751026.108374958.3653846932.8571429914.6929825884.1176471844.82456141003.5622321017.347826943.2894737900.7089552
CDilutionN/A1 in 41 in 41 in 4.51 in 4.51 in 51 in 51 in 5.51 in 5.51 in 61 in 5.51 in 6.51 in 5.5
OD0.2690.2390.2250.220.2030.240.2180.2250.2250.2310.2440.2360.237
YFP291.95243.21233.06207.05195.54221.06202.99193.67188.6233.191242.86219.04218.02
YFP/OD1085.3159851017.6150631035.822222941.1363636963.2512315921.0833333931.146789860.7555556838.22222221009.484848995.3278689928.1355932919.9156118
P85ADilutionN/A1 in 41 in 41 in 4.51 in 4.51 in 51 in 51 in 5.51 in 5.51 in 61 in 5.51 in 6.51 in 5.5
OD0.2210.2270.2180.20.1960.2320.2260.2120.2170.2390.2340.2180.221
YFP245.2236.07228.69202.98190.63220.43208.21184.71187.91239.59236.01212.75209.69
YFP/OD1109.5022621039.9559471049.0366971014.9972.6020408950.1293103921.2831858871.2735849865.94470051002.4686191008.589744975.9174312948.8235294
BDilutionN/A1 in 41 in 41 in 4.51 in 4.51 in 51 in 51 in 5.51 in 5.51 in 6.51 in 5.51 in 6.51 in 5.5
OD0.2210.2330.2190.2020.1920.2440.2230.2130.220.2290.2250.2250.225
YFP245.2232.52224.04200.66190.6220204.88181.33179.51233.89227.6211.73209.61
YFP/OD1109.502262997.93991421023.013699993.3663366992.7083333901.6393443918.7443946851.314554815.95454551021.3537121011.555556941.0222222931.6
CDilutionN/A1 in 41 in 41 in 4.51 in 4.51 in 51 in 51 in 5.51 in 5.51 in 61 in 61 in 6.51 in 5.5
OD0.2210.230.2260.20.1990.2380.2290.2160.2170.2380.2320.2340.227
YFP245.2233.88229.97199.54193.24215.08208.23182.57182.58234.45232.47217.81210.75
YFP/OD1109.5022621016.8695651017.566372997.7971.0552764903.697479909.30131845.2314815841.3824885985.08403361002.025862930.8119658928.4140969
(-) Ctrl (P41)ADilutionN/A1 in 41 in 41 in 4.51 in 4.51 in 51 in 51 in 5.51 in 5.51 in 61 in 5.51 in 6.51 in 5.5
OD0.2160.2320.2150.1970.1990.2370.2210.2170.2230.2340.2370.2260.234
YFP234.27227.67216.53186.19198.19214.86204.3190.44205.31236.74234.19211.12215.13
YFP/OD1084.583333981.33620691007.116279945.1269036995.9296482906.5822785924.4343891877.6036866920.67264571011.709402988.1434599934.159292919.3589744
BDilutionN/A1 in 41 in 41 in 4.51 in 4.51 in 51 in 51 in 5.51 in 5.51 in 61 in 61 in 6.51 in 5.5
OD0.2160.2270.2340.1990.1970.2350.2230.2170.2190.2420.2330.2250.228
YFP234.27237.44226.99190.07186.35215.29210.32190.44188.45239.24234.96209.83207.08
YFP/OD1084.5833331045.991189970.042735955.1256281945.9390863916.1276596943.1390135877.6036866860.5022831988.59504131008.412017932.5777778908.245614
CDilutionN/A1 in 41 in 41 in 4.51 in 4.51 in 51 in 51 in 5.51 in 5.51 in 61 in 61 in 6.51 in 5.5
OD0.2160.2190.2180.1930.1940.2380.2280.2140.2190.2410.2290.220.223
YFP234.27224.51227.38201.61190.81216.18209.61185.68183.13240.67227.78208.24207.36
YFP/OD1084.5833331025.1598171043.0275231044.611399983.556701908.3193277919.3421053867.6635514836.2100457998.6307054994.6724891946.5454545929.8654709


Data for the 1000fold Dilutions after 2 Hours

' ' ' Time ' ' ' ' ' ' ' ' '
2hrsDiluted 1000 fold, grown for 5 hours.Diluted 1000 fold, grown for 5 hours, diluted to same OD range.
StrainReplicate3030 + 1mM IPTG40423030 + 1mM IPTG4042* DID NOT SHAKE FOR TWO HOURS IN THE MIDDLE.4042* DID NOT SHAKE FOR TWO HOURS IN THE MIDDLE.
LBN/ADilutionN/AN/AN/AN/AN/AN/AN/AN/A1 in 101 in 10
OD0000000000
YFP40.7240.7240.7240.7246.846.846.846.846.846.8
P84ADilution1 in 41 in 41 in 4.51 in 4.5N/AN/AN/AN/A1 in 101 in 10
OD0.2290.2180.2160.2140.0180.0150.120.1390.0150.018
YFP242.89226.53216.85211.2163.5259.56164.97169.1559.2961.78
YFP/OD1060.6550221039.128441003.935185986.96261683528.8888893970.6666671374.751216.9064753952.6666673432.222222
BDilution1 in 41 in 41 in 4.51 in 4.5N/AN/AN/AN/A1 in 101 in 10
OD0.2210.2180.2030.2080.0150.0160.1340.1270.020.018
YFP225.35229.19208.3199.3462.1363.2169.54170.4961.662.48
YFP/OD1019.6832581051.3302751026.108374958.3653846414239501265.2238811342.44094530803471.111111
CDilution1 in 41 in 41 in 4.51 in 4.5N/AN/AN/AN/A1 in 101 in 10
OD0.2390.2250.220.2030.0170.0160.1310.1270.0210.016
YFP243.21233.06207.05195.5460.8662.2164.97163.959.7464.82
YFP/OD1017.6150631035.822222941.1363636963.251231535803887.51259.3129771290.5511812844.7619054051.25
P85ADilution1 in 41 in 41 in 4.51 in 4.5N/AN/AN/AN/A1 in 101 in 10
OD0.2270.2180.20.1960.0140.0170.1210.1220.0180.016
YFP236.07228.69202.98190.6359.2862.21155.08173.2963.3461.37
YFP/OD1039.9559471049.0366971014.9972.60204084234.2857143659.4117651281.6528931420.4098363518.8888893835.625
BDilution1 in 41 in 41 in 4.51 in 4.5N/AN/AN/AN/A1 in 101 in 10
OD0.2330.2190.2020.1920.0140.0120.1210.1140.0180.016
YFP232.52224.04200.66190.660.4756.56149.7417560.9957.54
YFP/OD997.93991421023.013699993.3663366992.70833334319.2857144713.3333331237.5206611535.0877193388.3333333596.25
CDilution1 in 41 in 41 in 4.51 in 4.5N/AN/AN/AN/A1 in 101 in 10
OD0.230.2260.20.1990.0150.0130.1260.1070.0210.016
YFP233.88229.97199.54193.2457.3158.69171.68148.3660.5863.76
YFP/OD1016.8695651017.566372997.7971.05527643820.6666674514.6153851362.5396831386.5420562884.7619053985
(-) Ctrl (P41)ADilution1 in 41 in 41 in 4.51 in 4.5N/AN/AN/AN/A1 in 101 in 10
OD0.2320.2150.1970.1990.0160.0150.1110.1330.0190.018
YFP227.67216.53186.19198.1965.6965.34150.96162.9460.4659.28
YFP/OD981.33620691007.116279945.1269036995.92964824105.625435613601225.1127823182.1052633293.333333
BDilution1 in 41 in 41 in 4.51 in 4.5N/AN/AN/AN/A1 in 201 in 10
OD0.2270.2340.1990.1970.0290.0270.2280.1250.0210.022
YFP237.44226.99190.07186.3571.7171.9241.84160.5759.661.7
YFP/OD1045.991189970.042735955.1256281945.93908632472.7586212662.9629631060.7017541284.562838.0952382804.545455
CDilution1 in 41 in 41 in 4.51 in 4.5N/AN/AN/AN/A1 in 101 in 10
OD0.2190.2180.1930.1940.0170.0180.1340.1180.0210.019
YFP224.51227.38201.61190.8159.5661.47167.86162.1459.5757.91
YFP/OD1025.1598171043.0275231044.611399983.5567013503.52941234151252.6865671374.0677972836.6666673047.894737

Sequencing Parts, Plasmids, Etc.

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

Primer Name Sequence
P4P13fwdGGATCTCGACGCTCTCCCTT
P12fwdATGCGTCCGGCGTAGAGGA
DuetRevTGCTAGTTATTGCTCAGCGG

These arrived 7/25/08, and were reconstituted to 100uM.

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

Gel Results 7/23

Image:7-23-08_analyticaldigA.jpg 1% Agarose, visualized using EtBr/UV
Lane Sample
11 kB ladder
2S1 P75a 1 (7/22)
3S1 P75a 2 (7/22)
4S1 P75b 1 (7/22)
5S1 P75b 2 (7/22)
6E1 P92b 1:1 (7/17)
7E1 P92a 1:1 (7/17)
8E1 P93a 1:1 (7/17)
9E1 P93b 1:1 (7/17)
10E1 P92a PCR (7/18)
11S1 P93 1:3 1 (7/17)
12S1 P93 1:3 2 (7/17)
Image:7-23 analyticdig B ls.jpg‎ 1% Agarose, visualized using EtBr/UV
Lane Sample
11 kB ladder
2S1 P92a PCR 1 (7/18)
3S1 P92a PCR 2 (7/18)
4S1 P93b PCR 1 (7/18)
5S1 P93b PCR 2 (7/18)
6E1 P58b 1 (7/18)
7E1 P58b 2 (7/18)
8S1 P58b A (7/15)
9S1 P58b B (7/15)
10S1 P59 (7/9)
11S1 P59b 1 (7/22)
12S1 P59b 2 (7/18)

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


Digestion Gel Results 7/23

Image:7-23 digest lts.jpg‎ 1% Agarose, visualized using EtBr/UV
Lane Sample
11kB Ladder
3S1 P75a (2750/925)
4100bp Ladder
5S1 P75b 1 (2750/925)
7P64a 07 (~3kb/129bp)
91kB Ladder

Extracted and purified S1 P75a 1 and P75b 1, but P64 07 has incorrect bands.

Desphosphorylated S1 P75a 1 and P75b 1 same day.

Digestion of (Almost) All of Our Terminators 7/23

' P41 P61 P62a 07 P64
DNA5 uL8 uL8 uL8 uL
100x BSA0.25 uL0.25 uL0.25 uL0.25 uL
10x EcoRI Buffer2.5 uL2.5 uL2.5 uL2.5 uL
Enzyme 11 uL EcoRI1 uL EcoRI1 uL EcoRI1 uL EcoRI
Enzyme 21 uL SpeI1 uL SpeI1 uL SpeI1 uL SpeI
Water15.25 uL 15.25 uL 15.25 uL 15.25 uL
Total25 uL25 uL25 uL25 uL

Gel? 7/24

Faint band was detected in P64 (?) but was not visible under UV when cutting, so not extracted. Will try digestion of P63, which has worked for Meng Xiao and Thilini before.

Ligation of P75 with old P63 7/24

Purified P63 cut with ES was ligated with the dephosphorylated P75 vector. Very little of the insert was available (~2 uL)

Transformed on Kan plates.

7/25: Almost all plates were blank except for positive control. One faint, questionable colony found on plate with 1 uL ligation reaction-- picked for Saturday.

Digestion of P63 (7/24) and E1 P75b (7/25)

' P63a P63b E1 P75b
DNA??10 uL
100x BSA0.25 uL0.25 uL0.25 uL
10x EcoRI Buffer2.5 uL2.5 uL2.5 uL
Enzyme 11 uL EcoRI1 uL EcoRI1 uL EcoRI
Enzyme 21 uL SpeI1 uL SpeI1 uL XbaI
Water??10.25 uL
Total25 uL25 uL25 uL

Gel Results 7/25

Image:7-25_digest_lts.jpg‎ 1% Agarose, visualized using EtBr/UV
Lane Sample
3E1 P75b (2750/ 925 bp)
51 kb Ladder
7P63a (95 bp)
8100 bp Ladder
9P63b (95 bp)

95 bp bands became fainter (and undetectable with gel mold on) after running the gel for more time.

E1 P75b, P63a, and P63b were extracted and purified. E1 P75b was dephosphorylated using the Roche T4 Ligation Kit Alkaline Phosphatase.

Ligation of pLambda+GFP Vector P75 with Terminator P63 7/25

' E1 P75b + P63a (6:2) E1 P75b + P63b (6:2) S1 P75a + P63a (6:2) S1 P75a + P63b (6:2) S1 P75b + P63a (6:2) S1 P75b + P63b (6:2) E1 P75b + P63a (5:1) E1 P75b + P63b (5:1) S1 P75a + P63a (5:1) S1 P75b + P63b (5:1)
Insert6 uL6 uL6 uL6 uL6 uL6 uL5 uL5 uL5 uL5 uL
Vector2 uL2 uL2 uL2 uL2 uL2 uL1 uL1 uL1 uL1 uL
DNA Dilution Buffer2 uL2 uL2 uL2 uL2 uL2 uL2 uL2 uL2 uL2 uL
Water (to 20 uL)0 uL0 uL0 uL0 uL0 uL0 uL2 uL2 uL2 uL2 uL
Ligation Buffer10 uL10 uL10 uL10 uL10 uL10 uL10 uL10 uL10 uL10 uL
Ligase1 uL1 uL1 uL1 uL1 uL1 uL1 uL1 uL1 uL1 uL

Transformation of P75+ P63 Ligations and PGW7

Plate Marker # Colonies Plate Description
E1 P75b + P63a (6:2)Kan0
E1 P75b + P63b (6:2)Kan0
S1 P75a + P63a (6:2)Kan1Single colony is fluorescent.
S1 P75a + P63b (6:2)Kan0
S1 P75b + P63a (6:2)Kan1Single colony NOT fluorescent.
S1 P75b + P63b (6:2)Kan0
E1 P75b + P63a (5:1)Kan0
E1 P75b + P63b (5:1)Kan0
S1 P75a + P63a (5:1)Kan0
S1 P75b + P63b (5:1)Kan0
PGW7 1 (1 uL plasmid in 50 uL cells)Amp200+
PGW7 2 (1 uL plasmid in 100 uL cells)Amp200+
puc19 30s HeatshockAmp18
puc19 45s HeatshockAmp15
puc19 1m HeatshockAmp6
puc19 2m HeatshockAmp14
Kan (-) ctrlKan0
Amp (-) ctrlAmp0

Gradient PCR of PGW7 Plasmid to Isolate cI857 7/25

Used primer sets RBS and BIG. Prepared enough for 4 standard reactions (50 uL each), and split into 8 reactions in strip top tubes.

Reaction Recipe:

Fwd Primer 1 uL
Rev Primer1 uL
Platinum Supermix45 uL
Water2 uL

Added a total of 2 uL of PGW7 plasmid to each reaction mix (0.25 uL plasmid per 25 uL reaction).

PCR Gels 7/25

PCR using RBS Primer Set:

  • Amplified region is cI857 coding region only, and primers have Biobricks prefix, suffix, and RBS.
  • 675bp
Image:7-25-08_cI857_RBS_al.jpg 1% Agarose, visualized using EtBr/UV
Lane Annealing Temperature
1100 bp Ladder
240.8
341.5
442.5
543.9
7100bp Ladder
845.4
946.7
1047.6
1148.3
131 kB Ladder

All bands were extracted and purified.

PCR using BIG Primer Set:

  • Amplified region is cI857 coding region and 200bp upstream and downstream, and primers have Biobricks prefix and suffix.
  • 1193bp
Image:7-25-08_cI857_BIG_al.jpg 1% Agarose, visualized using EtBr/UV
Lane Annealing Temperature
11 kB Ladder
240.8
341.5
442.5
543.9
645.4
746.7
847.6
948.3
10100 bp Ladder

All bands were extracted and purified.

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.

New cells seem to work, but tested heatshock times with puc19 7/25/08.


Ligations 07/22

  • 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)

Ligations 07/24

We ligated p75a w/ p63 w/ a ratio of 4ul p63:1ul p75a

Transformed into new dh5α competent cells w/ volumes of 1, 3, and 5uL, using Jason's new protocol. As a control transformed 1uL of pUC19.

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.

Lower gradient

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

7/23: gel

All the temperatures seem to have worked (51 was not loaded due to # lanes on gel). Expected product size ~2.1kb. Ladder is 1KB.

Products from different temperatures combined and gel purified.

Image:7-23_mtrB_nonBB_mxh.jpg

P51, 52, 17 MIT

17, 52:
Rx mix (split into 4 samples): 180μL Platinum PCR supermix, 4μL BBpfx primer (20μM), 4μL BBsfx primer (20μM), 4μL miniprepped DNA, 8μL water
51:
Rx mix (split into 4 samples): 100μL AmpliTaq Gold Master Mix, 4μL BBpfx primer (20μM), 4μL BBsfx primer (20μM), 4μL miniprepped DNA, 88μL water

Rx: 5min @ 94°C → 35x[45s @ 94°C → 45s @ {52.7, 53.6, 54.6, 55.5}°C → 1m25s @72°C] → 5min @ 72°C → ∞ @ 4°C

Results

Image:2008-07-23_pcr_mxh.jpg 1.2% agarose E-gel run for 30 min and visualized using EtBr/UV
Lane Contents
1-4P17 (902) (annealing temp increase LTR)
5-8P51 (1370) (annealing temp increase LTR)
9-11P53 (987) (annealing temp increase LTR)
121KB plus ladder

P1, P3 backbones; HO-pcyA; P51 MIT

Conditions optimization

Rx: 5min @ 94°C → 35x[45s @ 94°C → 45s @ {middle 8 of 41-55 gradient: 43.3, 44.9, 47.0, 49.3, 51.3, 52.8, 53.9, 54.7}°C → 3m30s @72°C] → 5min @ 72°C → ∞ @ 4°C

P1, P3

PCR product is the backbone of P1 and P3, including the RBS (B0032), weak and strong promoter respectively, and terminator. The ends have unique ApaLI and KpnI cut sites.

Rx mix (split into 8 samples): 180μL Platinum PCR supermix, 4μL P3minusGFP-F primer (20μM), 4μL P3minusGFP-R primer (20μM), 4μL miniprepped DNA (P1/P3), 8μL water

HO-pcyA

Ends have unique ApaLI and KpnI cut sites.

Rx mix (split into 8 samples): 180μL Platinum PCR supermix, 4μL HO-pcyA-F primer (20μM), 4μL HO-pcyA-R primer (20μM), 4μL miniprepped P86, 8μL water

P51 MIT

Rx mix (split into 4 samples): 90μL Platinum PCR supermix, 2μL BBsfx primer (20μM), 2μL BBpfx primer (20μM), 2μL miniprepped P51, 4μL water

Used highest 4 temps of gradient.

Gels

Annealing temp increases LTR.

Image:2008-07-24_pcr_1_mxh.jpg 1.2% agarose E-gel run for 30 min and visualized using EtBr/UV
Lane Contents
1-8P1 backbone (~3kb)
91 KB ladder
10-12HO-pcyA (~1.4 kb)
Image:2008-07-24_pcr_2_mxh.jpg 1.2% agarose E-gel run for 30 min and visualized using EtBr/UV
Lane Contents
1-5HO-pcyA (~1.4 kb)
61 KB ladder
7-12P3 backbone (~3 kb)
Image:2008-07-24_pcr_3_mxh.jpg 1.2% agarose E-gel run for 30 min and visualized using EtBr/UV
Lane Contents
1-2P3 backbone (~3kb)
31 KB ladder
4-7P51 MIT (~1.4 kb)

Full Rx

400 μL (8 reactions) each of P1 and P3 were set up using a doubling of above reaction mix. The reaction conditions were the same except that annealing temperature was 45°C, and cycles occur 40 times.

100 μL (2 reactions) each of HO-pcyA and P51 MIT were set up using above reaction ratios. 40 cycles were performed with 55°C annealing temp and 1:35 extension time.

Products will be gel purified along with products from optimization PCRs.

RE digests 07/22

Gel 1

Image:7-22_gel_1_MXHTA.jpg 1 agarose gel visualized using EtBr/UV
Lane Contents
11 kb plus ladder
2CDF PCR cut XP (~900)
3CDF PCR uncut (~900)
4P1A cut XP (2750)
5P1A uncut (3669)
6P17 cut EX (5327)
7P17 uncut (5327)
8P48 cut XP (769)
9P48 uncut (3477)

Gel 2

Image:7-22_gel_2_MXHTA.jpg 1 agarose gel visualized using EtBr/UV
Lane Contents
11 kb plus ladder
2P49B cut XP (943)
3P49B uncut (3651)
4P51 cut EX (5795)
5P51 uncut (5795)
6P52 cut EX (5412)
7P52 uncut (5412)
8P63 cut EX (3284)
9P63 uncut (3284)


Gel 3

Image:7-22_gel_3_MXHTA.jpg 1 agarose gel visualized using EtBr/UV
Lane Contents
11 kb plus ladder
2P97A cut EX (2091)
3P97A uncut (2091)
4P97B cut EX (2091)
5P97B uncut (2091)

Ligation w/ purified products 7/22: CDF into P1 vector

  • We ligated P1A (vector) to CDF. We used three different ratios of vector to insert for the ligation: 2/6, 1/5, 1/7.

RE digests 07/23

Gel 1: 1% agarose gel visualized using EtBr/UV Lane Contents
Image:7-23_gel_1_MXHTA.jpg 1 1 kb plus ladder
2 P97A cut SP (2091)
3 P97A uncut
4 P97B cut SP (2091)
5 P97B uncut
6 P51 cut EX (5795)
7 P51 uncut
8 P52 cut EX (5412)
9 P52 uncut
10 P90ζ cut SP (~3600)
11 P90ζ uncut
12 P90ε cut SP (~3600)
13 P90ε uncut
14 mtrB (not BioBrick) uncut PCR product (~2.1kb)
Gel 2: 1% agarose gel visualized using EtBr/UV Lane Contents
Image:7-23_gel_2_MXHTA.jpg 1 1 kb plus ladder
2 P17 cut EX (5327)
3 P17 uncut
4 S1P3A B cut XP (2750)
5 S1P3A B uncut
6 S1P3A C cut XP (2750)
7 S1P3A C uncut
8 P3A cut XP (2750)
9 P3A uncut
10 P3B cut XP (2750)
11 P3B uncut
12 mtrB BB cut XP (2100)
13 mtrB BB uncut

Ligations with purified digest fragments 07/23: mtrB+RBS, CDF into pSB3K3

We ligated

  • P97 and mtrB BB
  • P3 and CDF

We used three different ratios of insert to vector for the ligation: 6/2, 5/1, 7/1.

We used 5 μL of the ligation to transform TOP10 cells and 5 μL to transform the new DH5α cells. We also used 1 μL and 3 μL to transform DH5α. We used pUC19 (1 μL) as a positive control to test the efficacy of the new DH5α cells.

We used Jason's protocol to transform the new DH5α cells:

  • Thaw cells by resting tubes on ice
  • Then add DNA and mix by swirling with the pipette tip
  • Incubate the cells with DNA in ice for 10min
  • Heat shock at 42C for 2min
  • Then incubate in ice for 2-5min
  • Add 500-700ml LB (or SOC) and incubate + 250rpm shaking for 1hr at 37C
  • Plate

Results

Strain DNA Vector:Insert (μL) Amount transformed (μL) Plate # colonies
E1pUC19n/a1AMP40
E1-n/a-CARB0
E2P97+ mtrB BioBrick2:65CARB136
E2P97+ mtrB BioBrick1:55CARB48
E2P97+ mtrB BioBrick1:75CARB64
E1P97+ mtrB BioBrick1:71CARB0
E1P97+ mtrB BioBrick1:73CARB0
E1P97+ mtrB BioBrick1:75CARB4
E1P97+ mtrB BioBrick1:51CARB0
E1P97+ mtrB BioBrick1:51CARB0
E1P97+ mtrB BioBrick1:51CARB0
E1P97+ mtrB BioBrick2:61CARB1
E1P97+ mtrB BioBrick2:61CARB7
E1P97+ mtrB BioBrick2:61CARB9
E2P3+CDF2:65KAN7
E2P3+CDF1:55KAN56
E2P3+CDF1:75KAN5
E1P3+CDF1:71KAN0
E1P3+CDF1:73KAN2
E1P3+CDF1:75KAN0
E1P3+CDF1:51KAN0
E1P3+CDF1:53KAN0
E1P3+CDF1:55KAN0
E1P3+CDF2:61KAN0
E1P3+CDF2:63KAN1
E1P3+CDF2:65KAN0
  • Try 45s heat shock and SOC for new cells
  • Try 3 or 5 μL transformations
  • New Carb plates seem fine

RE digests of miniprepped ligations 07/25

Image:7-25_gel_1_MXHTA.jpg

Lane 1: 1 kb ladder

Lanes 2-7: P90 A-F cut SP (~3600)

Lanes 8-13: P98 A-F cut ES (~2100)

Lane 14: P3 cut XP (2750)

Ligations with purified parts and PCR products 07/26

Ligations were performed using standard protocol. New homemade cells were heat shocked for 40s, and incubated in 200μL SOC for 1 hour prior to plating on prewarmed plates. Vectors were dephosphorylated with Antarctic Phosphatase as per NEB's protocol.

Results 7/27
DNA Strain Vector,Insert Plate No. of Colonies
P3+HO-pcyA not BB PCRE13,5Kan1
P3+HO-pcyA not BB PCRE22,6Kan~50
P1+HO-pcyA not BB PCRE22,6Kan~50
P1+HO-pcyA not BB PCRE13,5Kan0
P1+mtrB not BB PCRE22,6Kan32
P1+mtrB not BB PCRE13,5Kan0
P3+mtrB not BB PCRE13,5Kan13
P3+mtrB not BB PCRE22,6Kan~80
P63+98E11,7AmpTMTC
P63+98E22,6AmpTMTC
P3+51 PCRE22,6Kan~120
P3+51 PCRE13,5Kan59
P90+49E11.5,6.5AmpTMTC
P90+49E22,6AmpTMTC
P90+48E11.5,6.5Cm~80
P90+48E22,6CmTMTC
pUC19 (positive control)E11 ulCarb~200

2 non-fluorescent colonies picked for 5mL cultures for each plate when possible.

RE digests 07/24

Image:7-24_gel_1_MXHTA.jpg 1% agarose gel visualized using EtBr/UV
Lane Contents
11 kb ladder
2P1A uncut (~3600)
3P1A cut EP (~2750)
4P3A uncut (~3600)
5P3A cut EP (~2750)
6P17 PCR cut XP (902)
7P52 PCR cut XP (987)
8P90.4 uncut (~3600)
9P90.1 cut SP (~3600)
10P90.2 cut SP (~3600)
11P90.3 cut SP (~3600)
12P90.4 cut SP (~3600)

Ligations w/ purified digest fragments 07/24

Vector:insert ratio: 2:6 μL, volume used in transformation: 4μL

Note that 45s heat shock was used for DH5α, and cells were split onto 2 plates to accelerate drying.

Results 7/25

DNA Strain Plate # Colonies
P1+P17E2KAN184
P1+P17E1KAN9
P1+P17E1KAN9
P1+P52E2KAN40
P1+P52E1KAN0
P1+P52E1KAN0
P3+P17E2KAN10
P3+P17E1KAN0
P3+P17E1KAN0
P3+P52E2KAN48
P3+P52E1KAN1
P3+P52E1KAN1
P38+P17E2KAN1
P38+P17E1KAN0
P38+P17E1KAN0
P38+P51E2KAN32
P38+P51E1KAN1
P38+P51E1KAN0
P38+P52E2KAN1
P38+P52E1KAN0
P38+P63E1KAN0
P39+P17E1KAN0
P39+P17E1KAN0
P39+P17E2KAN0
P39+P51E2KAN5
P39+P51E1KAN2
P39+P51E1KAN2
P39+P52E2KAN6
P39+P52E1KAN1
P39+P52E1KAN1
P90+P48E2CM16
P90+P48E1CM5
P90+P48E1CM1
P90+P49E2AMPTMTC
P90+P49E1AMP19
P90+P49E1AMP4
pUC19E1AMP192

Cultures for miniprep

When possible, 2 non-fluorescent colonies were picked from each plate (fluorescence in the P1/P3 derived plasmids indicates P1/P3 vector religation).

  • The 2 E2 P90+49 cultures failed to grow. Note that Kan selection was used, as that's carried on P90, although the transformation plates were Amp.

RE digests of minipreps 07/26

We digested

  • P46 with NheI
  • P90+49 and P90+48 with XbaI and SpeI
  • When these parts are combined, we will have a plasmid with the CDF origin (CDF+Amp and CDF+Cm in the base vector)
  • P3+17 and P1+17 with EcoRI and XbaI
  • We can add the high and low constitutive promoters to give us the complete Tet system (without GFP) in a p15A vector
  • P39+51, P38+51, P38+17 with XbaI and PstI
  • We can put these parts into P1 and P3 to give us the complete Tet and Lac systems in a p15A vector

Results

Gel 1: 1% agarose gel visualized using EtBr/UV Lane Contents (expected band sizes)
Image:2008-07-27_gel1_mxh.jpg
11KB PLUS LADDER
2E2 90+48A XS (1650+2750)
3E2 90+48B XS (1650+2750)
4E1 90+48A XS (1650+2750)
5E1 90+48B XS (1650+2750)
6E1 90+48C XS (1650+2750)
7E1 90+49A XS (1800+2750)
8E1 90+49B XS (1800+2750)
9E1 39+51A XP (1400+4425)
10E2 39+51B XP (1400+4425)
11E2 39+51A XP (1400+4425)
12E1 90+49D XS (1800+2750)
13E1 90+49C XS (1800+2750)
14E1 39+51B XP (1400+4425)
15E1 39+51C XP (1400+4425)
Gel 2: 1% agarose gel visualized using EtBr/UV Lane Contents (expected band sizes)
Image:2008-07-27_gel2_mxh.jpg
1E1 39+51D XP (1400+4425)
2E2 38+51A XP (1400+4425)
3E2 38+51B XP (1400+4425)
4E2 38+17 XP (950+2750)
5E1 38+51 XP (1400+4425)
6P46B NheI (943+1495)
7E1 1+17A EX
8E1 1+17B EX
9E1 1+17C EX
10E1 1+17D EX
11E2 1+17A EX
12E2 1+17B EX
13E2 3+17A EX
14E2 3+17B EX
15P1B XP (919+2750)
16P3A XP (919+2750)
171KB PLUS LADDER

Cultures were set up again for samples 7-14, since the DNA seems to have not run properly.

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