Revision as of 23:47, 19 October 2016

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 ---- DETAILS ON THIS NEEDED

Thursday, 8/18

Bryan flipped LB +AMP plates.

Monday, 8/29

Cloning Prep Calculations

Calculation for equamolar 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	Ingredient	amount
2	dH20	34uL
3	10x Cutsmart	5uL
4	NotI	1uL
5	100ng DNA	10uL

Restriction Digest for parts

Thursday, 9/1

Transformations

Chassi JM109

	A	B	C	D	E
1	Plate Number		Ratio (V:I)	Ligase presents (+/-)	Results (# of colonies)
2	1	Detector	1:1	+
3	2	Feedback	1:1	+
4	3	sfGFP	1:1	+
5	4	tnaA	1:1	+
6	5	Detector	1:2	+	1
7	6	Feedback	1:2	+
8	7	sfGFP	1:2	+
9	8	tnaA	1:2	+	1
10	9	Detector	1:1	-
11	10	Feedback	1:1	-
12	11	sfGFP	1:1	-
13	12	tnaA	1:1	-
14	13	Positive Control	PSBLC3	-	8

Table1

sfGFP- super folder green florecencent protien

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 innoculated 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 linerized PSB1C3 backbone.

	A	B	C
1		Part	Control tube
2	dH2O	17 uL	7 uL
3	10x Cutsmart Buffer	5 uL	1 uL
4	NotI (20U/uL)	1 uL	0.5 uL
5	DnpI (20U/uL)	1 uL	0.5 uL
6	rSAP	1 uL	--
7	total	25 uL	9 uL

Backbone Master Mix

4 uL of the above "Part" mixture and 4 uL of the 25ng/uL Linerized Backbone were mixed producing a DNA concentration of 12.5 ng/uL.

1 uL of the 25 ng/uL Linerized Backbone was added to the "Control tube" producing a DNA conacentration 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	Feedback	Detector	sfGFP
2	dH2O	11.5 uL	11.5 uL	11.5 uL	11.5 uL
3	10x cutsmart buffer	2.5 uL	2.5 uL	2.5 uL	2.5 uL
4	gBlock DNA (10ng/uL)	10 uL	10 uL	10 uL	10 uL
5	NotI-HF (20U/uL)	1 uL	1 uL	1 uL	1 uL
6	total volume	25uL	25uL	25uL	25uL

Parts Digest using Noti-HF

DNA now in concentation 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		tnaA	Feedback	Detector	sfGFP	control
2	dH2O	0.25 uL	0.25 uL	0.25 uL	0.25 uL	3uL
3	10x T4 Ligase Buffer	1 uL	1 uL	1 uL	1 uL	1.5 uL
4	Backbone mixture (12.5ng/uL)	2 uL	2 uL	2 uL	2 uL	---
5	Backbone mixture for control ( 2.5n/uL)	---	---	---	---	10 uL
6	Parts Digest (4ng/uL)	6.25 uL	6.25 uL	6.25 uL	6.25 uL	---
7	T4 ligase	0.5 uL	0.5 uL	0.5 uL	0.5 uL	0.5 uL
8	Total Volume	10 uL	10 uL	10 uL	10 uL	15 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 Mix	Control Mix
2	dH2O	17 uL	7 uL
3	10x Cutsmart Buffer	5uL	1 uL
4	Vector	--	1 uL
5	NotI (20U/uL)	1uL	0.5uL
6	DnPI(20U/uL)	1uL	0.5uL
7	rSAP	1 uL	--
8	total volume	25 uL	10 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		Detector	tnaA	Feedback	sfGFP	-Insert	Control Mix
2	dH2O	0.5 uL	0.5 uL	0.5 uL	0.5 uL	6.5 uL	3 uL
3	10x T4 ligase buffer	1 uL	1 uL	1 uL	1 uL	1 uL	1.5 uL
4	Vector (12.5ng/uL)	2 uL	2 uL	2 uL	2 uL	2 uL	10 uL
5	gBlock digest (4ng/uL)	6.25 uL	6.25 uL	6.25 uL	6.25 uL	--	--
6	T4 ligase	0.5 uL	0.5 uL	0.5 uL	0.5 uL	0.5 uL	0.5 uL
7	total volume	10 uL	10 uL	10 uL	10 uL	10 uL	15 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	dH20	18.5 uL	5.5 uL	5.5 uL
3	10x cutsmart buffer	2.5 uL	2.5 uL	2.5 uL
4	BBa_J04450	2 uL	10 uL	10 uL
5	NotI-HF (20u/uL)	1 uL	1 uL	1 uL
6	rSAP	1 uL	1 uL	1 uL
7	total	25 uL	25 uL	25 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

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 Insert	Vector Only
2	dH2O	3.5 uL	19.5 uL
3	10x T4 ligase buffer	2.5 uL	2.5 uL
4	Vector (10ng/uL)	3.0 uL	3.0 uL
5	gBlock digest (4ng/uL)	15 uL	--
6	T4 ligase	1 uL	1 uL
7	total volume	25 uL	25 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	dH20	2.5 uL
3	10x cutsmart buffer	1.5 uL
4	Detector gBlock (10ng/uL)	10 uL
5	NotI-HF (20u/uL)	1 uL
6	total	15 uL
7	Final Concentration	6.67 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		Feedback	Vector Only
2	dH2O	3.5 uL	18.5 uL
3	10x T4 ligase buffer	2.5 uL	2.5 uL
4	Vector (10ng/uL)	3.0 uL	3.0 uL
5	gBlock digest (4ng/uL)	15 uL	--
6	T4 ligase	1 uL	1 uL
7	total volume	25 uL	25 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 Mix	Tc	Volts
2	1	0.5 pg/uL control BBa_J04450	4.8 ms	1785 V
3	2	10 pg/uL control BBa_J04450	4.9 ms	1786 V
4	3	Vector Only Ligation	4.7 ms	1785 V
5	4	Detector Ligation	4.4 ms	1783 V
6	5	Cells only	5.0 ms	1785 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

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 Plasmid	2 ug Plasmid
2	dH20	37 uL	29 uL
3	10x cutsmart buffer	5 uL	5 uL
4	BBa_J04450	7 uL	14 uL
5	NotI-HF (20u/uL)	1 uL	2 uL
6	total	50 uL	50 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		Detector	tnaA	Feedback
2	dH20	24 uL	24 uL	24 uL
3	10x Cutsmart Buffer	5 uL	5 uL	5 uL
4	gBlock	20uL	20uL	20uL
5	NotI-HF	1 uL	1 uL	1 uL
6	total volume	50 uL	50 uL	50 uL

Table10

Ran at 37°C for one hour, then concentrated using vacum 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

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 Vector	Feedback, 9/30 Vector	Detector, 10-15 Vector	Detector, 9/30 Vector	10-15 Vector Only	9/30 Vector Only
2	dH2O	9 uL	9 uL	9 uL	9 uL	24 uL	24 uL
3	10x T4 ligase buffer	5 uL	5 uL	5 uL	5 uL	5 uL	5 uL
4	Vector	20 uL	20 uL	20 uL	20 uL	20 uL	20 uL
5	gBlock digest	15 uL	15 uL	15 uL	15 uL	--	--
6	T4 ligase	1 uL	1 uL	1 uL	1 uL	1 uL	1 uL
7	total volume	50uL	50uL	50uL	50uL	50uL	50uL

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	1	18	2.5	2.5	1	1	25
3	2	17	2.5	3.5	1	1	25
4	3	16.35	2.5	4	1	1	25
5	4	12.5	2.5	8	1	1	25
6	5	9.5	2.5	11	1	1	25
7	6	11.5	2.5	9	1	1	25
8	7	6.5	2.5	14	1	1	25
9	8	5.5	2.5	15	1	1	25
10	9	10	2.5	10.5	1	1	25

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.

@@ Line 52: / Line 52: @@
 </body>
 </html>
+{{SCSU-New_Haven/Footer}}

Difference between revisions of "Team:SCSU-New Haven/Notebook"

Revision as of 23:47, 19 October 2016

SCSU iGEM Project work

10-1_iGEM_gel_pure_2016.10.01_10.14.15_Fl-UV.jpg

10-6_iGEMgel_ligmixs_2016.10.06_15.06.55_Fl-UV.jpg

10-14 VectorDigest 2016.10.14_15.18.11_Fl-UV.jpg