Team:SCSU-New Haven/Notebook

SCSU iGEM Project work · Benchling

SCSU iGEM Project work

Made with Benchling
Project: SCSU_NHiGEM Shared Project
Authors: Bryan Pasqualucci
Dates: 2016-08-17 to 2016-10-19
Wednesday, 8/17
Thomas made 1 liter of LB +AMP for solid media and 1 liter of LB liquid stock (AMP to be added later) according to protocol listed as "Media Preparation."
Poured LB +AMP plates, will flip plates on 8/18
Thomas and Dr. E made AMP stock
Thursday, 8/18
Bryan flipped LB +AMP plates.
Monday, 8/29
Cloning Prep Calculations
Calculation for equimolar amount of digested DNA in ligation step
1.
Digestion of linear backbone PSB1C3 using protocol Cloning Digest. Produced 4 tubes containing 100ng of digested backbone in 8 uL.
2.
Resuspended gBlocks parts , "tnaA V3", "Feedback V3", "Detector" and sfGFP reporter".
a.
gBlocks are 1000ng,resuspend in 100 uL of dH2O for 10 ng/uL
3.
Set up Restriction Digest for parts
a.
A
B
1
Ingredientamount
2
dH2034uL
3
10x Cutsmart5uL
4
NotI1uL
5
100ng DNA10uL
Restriction Digest for parts
Thursday, 9/1
Transformations
Chassis JM109
A
B
C
D
E
1
Plate NumberRatio (V:I)Ligase presents (+/-)Results (# of colonies)
2
1Detector1:1+
3
2Feedback1:1+
4
3sfGFP1:1+
5
4tnaA1:1+
6
5Detector1:2+1
7
6Feedback1:2+
8
7sfGFP1:2+
9
8tnaA1:2+1
10
9Detector1:1-
11
10Feedback1:1-
12
11sfGFP1:1-
13
12tnaA1:1-
14
13Positive ControlPSBLC3-8
Table1
sfGFP- super folder green florescent protein
tnnA- Tryptophanase gene
RESULTS:
Patched out suspected colonies from plates 5 and 8 to grow at 37 overnight.
Friday, 9/2
Patch on plate 8 grew overnight.
Suspected Colonies on plates 5 and 13 were picked and patched onto fresh LB+chloro plates.
Saturday, 9/3
No colonies grew on plate 5 patch.
Colonies from plate 13 and 8 were picked and inoculated into liquid LB+chloro and let grow overnight in 37 degrees C.
---Further work on this moved to tnaA
Saturday, 9/17
Bryan made Master digestion mixture for the linearized PSB1C3 backbone.
A
B
C
1
PartControl tube
2
dH2O17 uL7 uL
3
10x Cutsmart Buffer 5 uL1 uL
4
NotI (20U/uL)1 uL0.5 uL
5
DnpI (20U/uL)1 uL0.5 uL
6
rSAP1 uL--
7
total25 uL9 uL
Backbone Master Mix
4 uL of the above "Part" mixture and 4 uL of the 25ng/uL linearized Backbone were mixed producing a DNA concentration of 12.5 ng/uL.
1 uL of the 25 ng/uL Linearized Backbone was added to the "Control tube" producing a DNA concentration of 2.5 ng/uL.
These were incubated at 37°C for 30 minutes, then 80°C for 20 minutes to heat kill, then brought to 4°C until ready to ligate.
Bryan made Digests for each of the parts.
A
B
C
D
E
1
tnaA FeedbackDetectorsfGFP
2
dH2O11.5 uL11.5 uL11.5 uL11.5 uL
3
10x cutsmart buffer2.5 uL2.5 uL2.5 uL2.5 uL
4
gBlock DNA (10ng/uL)10 uL10 uL10 uL10 uL
5
NotI-HF (20U/uL)1 uL1 uL1 uL1 uL
6
total volume25uL25uL25uL25uL
Parts Digest using Noti-HF
DNA now in concentration of 4ng/uL.
Reactions were run at 37°C for 30 minutes then heat killed for 20 minutes at 80°C, then brought to 4°C until ligation step.
A
B
C
D
E
F
1
tnaAFeedbackDetectorsfGFPcontrol
2
dH2O0.25 uL0.25 uL0.25 uL0.25 uL3uL
3
10x T4 Ligase Buffer1 uL1 uL1 uL1 uL1.5 uL
4
Backbone mixture (12.5ng/uL)2 uL2 uL2 uL2 uL---
5
Backbone mixture for control ( 2.5n/uL)------------10 uL
6
Parts Digest (4ng/uL)6.25 uL6.25 uL6.25 uL6.25 uL---
7
T4 ligase0.5 uL0.5 uL0.5 uL0.5 uL0.5 uL
8
Total Volume10 uL10 uL10 uL10 uL15 uL
Table2
Ligation Reaction run overnight at 16°C (about 20 hours) then heat killed at 80°C for 20 minutes.
Sunday, 9/18
Mixtures were transformed using Transformation JM109 including the SOC outgrowth step and plated on LB+chloro at 37°C.
Monday, 9/19
BP Checked Plates and found no growth. Check transformation protocol, transform with PSB1C3 miniprep DNA next time. Try different plates, there may have been a problem with the Chloro.
May need to test all the chloro plates to find ones that aren't terrible if that is the problem.
SO could be problems : Transformation, plates, design is horribly messed up.
It's not : The enzymes not working, the ligase.
Tuesday, 9/20
Made Enzyme Master mix with rSAP
A
B
C
1
Master MixControl Mix
2
dH2O17 uL7 uL
3
10x Cutsmart Buffer5uL1 uL
4
Vector--1 uL
5
NotI (20U/uL)1uL0.5uL
6
DnPI(20U/uL)1uL0.5uL
7
rSAP1 uL--
8
total volume25 uL10 uL
Table3
From "Master Mix" 4uL was added to 4uL linear PSB1C3 and 4uL was added to 4uL of each part gBlock
Ran at 37°C for 30 minutes, then to 80°C for 20 minutes to heat kill, then to 4°C until next use.
Thursday, 9/22
The following Ligation reaction was set up with the previous day's digests.
A
B
C
D
E
F
G
1
DetectortnaAFeedbacksfGFP-InsertControl Mix
2
dH2O0.5 uL0.5 uL0.5 uL0.5 uL6.5 uL3 uL
3
10x T4 ligase buffer1 uL1 uL1 uL1 uL1 uL1.5 uL
4
Vector (12.5ng/uL)2 uL2 uL2 uL2 uL2 uL10 uL
5
gBlock digest (4ng/uL)6.25 uL6.25 uL6.25 uL6.25 uL----
6
T4 ligase0.5 uL0.5 uL0.5 uL0.5 uL0.5 uL0.5 uL
7
total volume10 uL10 uL10 uL10 uL10 uL15 uL
Table4
Reactions run at 16°C overnight, heat killed at 65°C for 20 minutes.
Friday, 9/23
Transformed previous day's ligations using protocol Transformation JM109 with SOC step.
Also transformed 1uL 10pg/uL of J04450 Plasmid from competent cell test kit.
Saturday, 9/24
Figured out why we were having such problems. The liner backbones that are provided have a messed up NotI site due to the way they were PCR produced. So because we have been trying to clone with NotI because of the stuffer sequence problem problems abound. We should break out BBa_J04450 and digest it ourselves with NotI, gel purify it and then try cloning with that.
Broke out BBa_J04450 from the micro wellplate distribution and Transformed it using Transformation JM109
Sunday, 9/25
Streaked out the transformed cells
Wednesday, 9/28
Picked single colonies into two 5mL LB+Chloro liquid cultures, set shaking at 250rpm at 37°C overnight
Thursday, 9/29
Performed miniprep's on the two cultures using Quigen QuickLyse MiniPrep kit (cat. 27406)
Measured Plasmid concentration using NanoDrop
#1 : 119.3 ng/uL
#2 : 23.33 ng/uL
Set up the following digestion
A
B
C
D
1
#1 (119.3ng/uL)#1-2 (119.3 ng/uL)#2 (23.33ng/uL)
2
dH2018.5 uL5.5 uL5.5 uL
3
10x cutsmart buffer2.5 uL2.5 uL2.5 uL
4
BBa_J044502 uL10 uL10 uL
5
NotI-HF (20u/uL)1 uL1 uL1 uL
6
rSAP1 uL1 uL1 uL
7
total25 uL25 uL25 uL
Table5
Ran at 37°C for 30 minutes, then to 80°C for 20 minutes to heat kill, then to 4°C until next use.
Ran on Gel at 10V overnight (20 hours)
Friday, 9/30
10-1_iGEM_gel_pure_2016.10.01_10.14.15_Fl-UV.jpg
thumbnail
From left to right (2-log ladder, #1-2 , #1, #2)
Extracted the #1-2 and #2 bands using Quigen Gel Extraction Kit (cat. 28706)
Combined the elutions and quantified the DNA using NanoDrop . 10ng/uL in 58uL solution.
Preformed a ligation reaction using the insert digests from 9-17.
A
B
C
1
1:3 V to InsertVector Only
2
dH2O3.5 uL19.5 uL
3
10x T4 ligase buffer2.5 uL2.5 uL
4
Vector (10ng/uL)3.0 uL3.0 uL
5
gBlock digest (4ng/uL)15 uL--
6
T4 ligase1 uL1 uL
7
total volume25 uL25 uL
Table6
Ligation ran at 16°C for 22 hours, Heat kill at 65°C for 10 minutes.
Saturday, 10/1
Transformed the previous ligation reactions using Transformation JM109
Plated and incubated at 37°C.
Sunday, 10/2
Checked plates, no colonies.
Going to redo the Insert Digestion, maybe we shouldn't have used the old digest?
A
B
1
Detector
2
dH202.5 uL
3
10x cutsmart buffer1.5 uL
4
Detector gBlock (10ng/uL)10 uL
5
NotI-HF (20u/uL)1 uL
6
total15 uL
7
Final Concentration6.67 ng/uL
Table7
Ran at 37°C for 30 minutes, then to 80°C for 20 minutes to heat kill, then to 4°C until next use.
Monday, 10/3
Ligation of 10-3 Digest
A
B
C
1
FeedbackVector Only
2
dH2O3.5 uL18.5 uL
3
10x T4 ligase buffer2.5 uL2.5 uL
4
Vector (10ng/uL)3.0 uL3.0 uL
5
gBlock digest (4ng/uL)15 uL--
6
T4 ligase1 uL1 uL
7
total volume25 uL25 uL
Table8
Ligation ran at 16°C overnight, Heat kill at 65°C for 10 minutes.
Tuesday, 10/4
Transformed ligation mixes into NEB electrocompetent cells (cat. C2989K) using BioRad Gene Pulser Xcell Electroporation System using "Bacterial 1" setting.
A
B
C
D
1
Cuvette #Ligation MixTc Volts
2
10.5 pg/uL control BBa_J044504.8 ms1785 V
3
210 pg/uL control BBa_J044504.9 ms1786 V
4
3Vector Only Ligation4.7 ms1785 V
5
4Detector Ligation4.4 ms1783 V
6
5Cells only5.0 ms1785 V
7
Table9
Plated onto LB+Chloro plates and incubated at 37°C
Wednesday, 10/5
Checked plates.
Only Control plates had growth.
Thursday, 10/6
Ran mixes with 2-log ladder on a Gel at 100V for one hour.
10-6_iGEMgel_ligmixs_2016.10.06_15.06.55_Fl-UV.jpg
thumbnail
It looks like almost nothing is there.... So concentration issues?
Going to redo the BBa_J04450 digest so we can get more vector.
See about concentration the inserts?
Tuesday, 10/11
Made two 5mL LB+Chloro cultures of BBa_J04450 from colonies isolated on 9/25.
Wednesday, 10/12
Performed miniprep's on the two cultures using Quigen QuickLyse MiniPrep kit (cat. 27406)
Did two preps per 5mL culture.
Combined the preps into 156.6 ng/uL in 196uL.
Thursday, 10/13
Digested the Plasmid from 10/12.
A
B
C
1
1 ug Plasmid2 ug Plasmid
2
dH2037 uL29 uL
3
10x cutsmart buffer5 uL5 uL
4
BBa_J044507 uL14 uL
5
NotI-HF (20u/uL)1 uL2 uL
6
total50 uL50 uL
Table11
Ran at 37°C for one hour, then to 80°C for 20 minutes to heat kill, then to 4°C until next use.
Friday, 10/14
Digested inserts then will run on Gel
A
B
C
D
1
DetectortnaAFeedback
2
dH2024 uL24 uL24 uL
3
10x Cutsmart Buffer5 uL5 uL5 uL
4
gBlock 20uL20uL20uL
5
NotI-HF1 uL1 uL1 uL
6
total volume50 uL50 uL50 uL
Table10
Ran at 37°C for one hour, then concentrated using vacuum centrifuge until volume was 20uL.
Ran 20uL of each of the Detector and Feedback gBlock inserts on a Gel to purify. Along with The 1ug and 2 ug Vector Digestions.
Ran at 10V overnight.
Saturday, 10/15
10-14 VectorDigest 2016.10.14_15.18.11_Fl-UV.jpg
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From left to right (2-log ladder, 1ug Vector digest, 2ug Vector Digest, Detector 2wells, Feedback 2 wells)
Extracted the desired bands using Quigen Gel Extraction Kit (cat. 28706)
NanoDrop Results
PSB1C3#1_10-15-16 Default 10/15/2016 11:53 AM 6.453 ng/ul 0.129 0.101 1.28 0.03 50.00 260 0.126 -0.018 DNA-50
PSB1C3#2_10-15-16 Default 10/15/2016 11:54 AM 9.759 ng/ul 0.195 0.144 1.36 0.04 50.00 260 0.191 0.027 DNA-50
Detector#1_10-15-16 Default 10/15/2016 11:55 AM 7.265 ng/ul 0.145 0.105 1.38 0.02 50.00 260 0.141 0.006 DNA-50
Detector#2_10-15-16 Default 10/15/2016 11:58 AM 6.098 ng/ul 0.122 0.100 1.22 0.03 50.00 260 0.118 -0.027 DNA-50
Feedback#1_10-15-16 Default 10/15/2016 11:59 AM 10.02 ng/ul 0.200 0.143 1.41 0.03 50.00 260 0.196 0.086 DNA-50
Feedback#2_10-15-16 Default 10/15/2016 12:00 PM3.931 ng/ul 0.079 0.071 1.11 0.08 50.00 260 0.076 0.031 DNA-50
Sunday, 10/16
Set up a Ligation Reaction using previous day's purifications
A
B
C
D
E
F
G
1
Feedback, 10-15 VectorFeedback, 9/30 VectorDetector, 10-15 VectorDetector, 9/30 Vector10-15 Vector Only9/30 Vector Only
2
dH2O9 uL9 uL9 uL9 uL24 uL24 uL
3
10x T4 ligase buffer5 uL5 uL5 uL5 uL5 uL5 uL
4
Vector20 uL20 uL20 uL20 uL20 uL20 uL
5
gBlock digest 15 uL15 uL15 uL15 uL----
6
T4 ligase1 uL1 uL1 uL1 uL1 uL1 uL
7
total volume50uL50uL50uL50uL50uL50uL
Table12
Incubated at Room temp (25°C for 10 minutes) and transformed using NEB DH5 alpha cells (cat. C2987I)
Plated onto LB+Chloro at 37°C and said a little prayer.
Monday, 10/17
COLONIESSSSS!!!
Those using the 10-15 Vector made red colonies meaning BBa_J04450 didn't get cut out all the way.
But the Vector Only from 9/30 produced very few colonies (2) and the Feedback and Detector produced 9 and 7 respectively.
Picked the 9 colonies from the Feedback plate and made patches and inoculated each into 5mL liquid LB+Chloro, 250rpm at 37°C.
Tuesday, 10/18
Performed two miniprep's per culture using Quigen QuickLyse MiniPrep kit (cat. 27406)
Quantified on the NanoDrop.
Sample ID User ID Date Time Conc. Units A260 A280 260/280 260/230 Conc. Factor (ng/ul) Cursor Pos. Cursor abs. 340 raw NA Type
1-1 Default 10/18/2016 3:08 PM 534.8 ng/ul 10.696 5.249 2.04 1.47 50.00 260 10.668 0.048 DNA-50
1-2 Default 10/18/2016 3:09 PM 308.3 ng/ul 6.165 2.983 2.07 1.29 50.00 260 6.143 0.032 DNA-50
2-1 Default 10/18/2016 3:10 PM 381.4 ng/ul 7.628 3.719 2.05 1.25 50.00 260 7.607 0.007 DNA-50
2-2 Default 10/18/2016 3:11 PM 224.2 ng/ul 4.484 2.180 2.06 1.23 50.00 260 4.468 -0.007 DNA-50
3-1 Default 10/18/2016 3:12 PM 201.5 ng/ul 4.031 2.020 2.00 1.08 50.00 260 4.014 -0.020 DNA-50
3-2 Default 10/18/2016 3:13 PM 316.3 ng/ul 6.325 3.080 2.05 1.16 50.00 260 6.304 -0.008 DNA-50
4-1 Default 10/18/2016 3:14 PM 75.87 ng/ul 1.517 0.793 1.91 0.81 50.00 260 1.508 -0.050 DNA-50
4-2 Default 10/18/2016 3:15 PM 171.8 ng/ul 3.436 1.726 1.99 1.06 50.00 260 3.422 -0.026 DNA-50
5-1 Default 10/18/2016 3:15 PM 96.04 ng/ul 1.921 0.994 1.93 0.91 50.00 260 1.911 -0.254 DNA-50
5-2 Default 10/18/2016 3:16 PM 87.27 ng/ul 1.745 0.900 1.94 0.93 50.00 260 1.735 -0.011 DNA-50
6-1 Default 10/18/2016 3:17 PM 143.7 ng/ul 2.874 1.449 1.98 1.13 50.00 260 2.860 -0.034 DNA-50
6-2 Default 10/18/2016 3:18 PM 79.38 ng/ul 1.588 0.797 1.99 0.93 50.00 260 1.578 -0.046 DNA-50
7-1 Default 10/18/2016 3:19 PM 75.08 ng/ul 1.502 0.778 1.93 0.80 50.00 260 1.493 -0.032 DNA-50
7-2 Default 10/18/2016 3:20 PM 68.95 ng/ul 1.379 0.720 1.91 0.84 50.00 260 1.372 -0.044 DNA-50
8-1 Default 10/18/2016 3:21 PM 84.03 ng/ul 1.681 0.863 1.95 0.80 50.00 260 1.671 -0.009 DNA-50
8-2 Default 10/18/2016 3:22 PM 52.02 ng/ul 1.040 0.544 1.91 0.78 50.00 260 1.033 -0.025 DNA-50
9-1 Default 10/18/2016 3:22 PM 70.20 ng/ul 1.404 0.732 1.92 0.82 50.00 260 1.396 -0.021 DNA-50
9-2 Default 10/18/2016 3:23 PM 122.6 ng/ul 2.451 1.249 1.96 1.03 50.00 260 2.440 -0.053 DNA-50
dh20 Default 10/18/2016 3:24 PM 0.3232 ng/ul 0.006 0.002 3.20 0.17 50.00 260 0.007 -0.071 DNA-50
Combined each culture's prep (ie 1-1 and 1-2 were combined, etc)
Set up the following Digestion to determine insert presence and direction.
A
B
C
D
E
F
G
1
Tube #dH2O (uL)10x cutsmart Buffer (uL)DNA approx 1 ug (uL)EcoRI (uL)SpeI (uL)total volume (uL)
2
1182.52.51125
3
2172.53.51125
4
316.352.541125
5
412.52.581125
6
59.52.5111125
7
611.52.591125
8
76.52.5141125
9
85.52.5151125
10
9102.510.51125
Table13
Digested at 37°C for 2 hours, 80°C for 20 minutes, then to 4°C until next use.
Wednesday, 10/19
Run them on a Gel and see Results. Gel shows 2kb Vector band and 1.8kb Insert band, in and correct!
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