Team:MIT/miRNA Group Notebook Experiment 2 3 miscellaneous

• BACKGROUND
• EXPERIMENTS
  Synthetic Promoters microRNA Recombinase Proof of Concept Collaborations
• FUTURE WORK
  Circuit Design Clinical Applications
• HUMAN PRACTICES
  Parts Toolbox Accessible Protocols Integrated Human Practices Public Outreach Interlab Study
• NOTEBOOK
  Lab Notebook Parts Mammalian Parts Collection Attributions Safety
• AWARDS
  HP Silver HP Gold Integrated HP Basic Part
• TEAM

Confirming FF4 Midiprep

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Elizabeth Strand

Date: 2016-09-02

Friday, 9/2

A: Confirms FF4 there (PacI in FF4 x6)

	A	B	C	D	E	F
1		Uncut (7.2)	PacI (7.2)	SpeI-HF (7.2)	Both (7.2)	Both (7.1)
2	DNA (~120 ng/ul)	3	3	3	3	3
3	10x Cutsmart	1	1	1	1	1
4	PacI	0	1	0	1	1
5	SpeI-HF	0	0	1	1	1
6	Nuclease free water	6	5	5	4	4
7	total volume	10	10	10	10	10

Table5

B: Confirms LacZ not there (SphI in LacZ ONLY)

	A	B	C	D	E
1		HpaI	SphI-HF (7.2)	Both (7.2)	Both (7.1)
2	DNA (~120 ng/ul)	3	3	3	3
3	10x Cutsmart	1	1	1	1
4	HpaI	1	0	1	1
5	SphI	0	1	1	1
6	Nuclease free water	5	5	4	4
7	total volume	10	10	10	10

Table1

Incubated at 37C 3:10pm-4:45pm

gel: Ladder, table A (in order), skip, Ladder, Ladder, table B (in order), skip, uncut

Golden Gate to form ERE3/5

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Elizabeth Strand

Date: 2016-10-09

Sunday, 10/9

	A	B	C	D	E	F	G
1	Part	Nickname	Size (kb)	Concentration, (ng/ul)	Concentration, (fmol/ul)	Volume for 50 fmol	Total
2	pERE5:TP901_GG-1	3-1	5.16	92.8	27.2	1.8
3	pERE5:mKate_GG-1	5-1	4.409	96.9	33.3	1.5
4	451a ultramer		0.136	1.72	19.2	2.6	10.4372093023
5	pERE3:TP901_GG-2	2-2	5.075	121	36.1	1.4
6	pERE3:mKate_GG-2	2-4	4.324	120.1	42.1	1.2

Table1

Record the reaction setup for each reaction below. For each reaction, include:

-- 50 fmol of each part

-- 1.5 ul T4 ligase buffer

-- 1.5 ul 10X BSA

-- 1 ul BbsI

-- 1 ul T4 ligase

-- Water to a total volume of 15 ul.

	A	B	C	D	E
1		"3-1"	"5-1"	"2-2"	4-2
2	Water	5.56	5.89	6.01	6.20
3	EREX:TP901 or mkate (100 ng/ul)	1.83	1.50	1.38	1.19
4	451a-ultramer (1.7 ng/ul)	2.61	2.61	2.61	2.61
5	T4 ligase buffer	1.50	1.50	1.50	1.50
6	10X BSA	1.50	1.50	1.50	1.50
7	BbsI ( 5k U/ml)	1.00	1.00	1.00	1.00
8	T4 ligase (2 mil U/ml)	1.00	1.00	1.00	1.00

Table2

Golden Gate to form pERE5/6_TP901_451

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Elizabeth Strand

Dates: 2016-10-04 to 2016-10-09

Tuesday, 10/4

Trying to form pERE5(or 6):TP901 with 451a target sit attached

	A	B	C	D	E
1	Part	Size (kb)	Concentration, (ng/ul)	Concentration, (fmol/ul)	Volume for 50 fmol
2	pERE5:TP901_GG	3.4	100	44.6	1.1
3	pERE6:TP901_GG	4.487	100	33.8	1.5
4	451a ultramer	0.136	1.72	19.2	2.6
5					5.2120123256

Table1

Record the reaction setup for each reaction below. For each reaction, include:

-- 50 fmol of each part

-- 1.5 ul T4 ligase buffer

-- 1.5 ul 10X BSA

-- 1 ul BsaI

-- 1 ul T4 ligase

-- Water to a total volume of 15 ul.

-- NOTE: If you're using AarI, you need to add the AarI oligo as well!

	A	B	C	D
1		"5"	"6"	"NEG"
2	Water	6.27	5.91	8.52
3	EREX:TP901 (100 ng/ul)	1.12	1.48	1.48
4	451a-ultramer (1.7 ng/ul)	2.61	2.61	0.00
5	T4 ligase buffer	1.50	1.50	1.50
6	10X BSA	1.50	1.50	1.50
7	BbsI	1.00	1.00	1.00
8	T4 ligase	1.00	1.00	1.00

Table2

	A	B
1	Heat lid	70°
2	Start cycle	15X
3	--	37° for 1' 30"
4	--	16° for 3' 0"
5	Close Cycle
6	50° for 5'
7	80° for 10'
8	Store at 8°

Table3

Transformations

pUC19 -- positive control

"5" = pERE5:TP901-451a --> 10 ul / 200 ul water

"6" = pERE6:TP901-451a --> 10 ul / 200 ul water

i4 = ? --> Regular (100 ul transformation)

	A	B	C	D
1	DNA	Description	Plates	Plating
2	i4	?	Kan + Xgal	5 ul + 200 ul H2o
3	pUC19	positive control	Kan+Xgal	100 ul
4	5	pERE5:TP901-451a	Amp + Xgal	10 ul + 200 ul H2O
5	6	pERE6:TP901-451a	Amp + Xgal	10 ul + 200 ul H2O
6	Neg	Negative control, pERE6:TP901 alone	Amp + Xgal	10 ul + 200 ul H2O
7	6	pERE6:TP901-451a	Amp + Xgal	5 ul + 200 ul H2O
8

Table4

Sunday, 10/9

Used similar protocol to do golden gate for ere3/5-tp901-451a

Jeremy miRNA Sensors

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Elizabeth Strand

Date: 2016-08-04

Thursday, 8/4

*Aliquots of miRNA sensors were given to us by Jeremy Gam in about 3uL aliquots, around 500ng/uL. 5uL dilutions were made to achieve 100ng/uL. Dilutions actually ranged 50ng/uL to 200ng/uL.

DNA was transformed into E. Coli and grown on Amp+Kan plates. Liquid cultures grown with amp.

Sensors Amplified:

miRNA-9 (mini= midi=

miRNA-451 (mini= midi=

miRNA-21 (mini= midi=

miRNA-20a (mini= midi=

miRNA-18a (mini= midi=

miRNA-200b (mini= midi=

miRNA-142 (mini= midi=

miRNA- (mini= midi=

miRNA- (mini= midi=

Restriction Digest:

BamHI and HpaI

	A	B	C	D	E	F
1	construct	enzymes	H20 (uL)	Enzyme (uL)	CutSmart (uL)	DNA (uL)
2	182	none	7	0	1	3
3	182	HpaI	6	1	1	3
4	182	BamHI	6	1	1	3
5	182	HpaI and BamHI	5	2	1	3
6		HpaI and BamHI	5	2	1	3
7		HpaI and BamHI	5	2	1	3
8		HpaI and BamHI	5	2	1	3
9		HpaI and BamHI	5	2	1	3
10		HpaI and BamHI	5	2	1	3
11		HpaI and BamHI	5	2	1	3
12		HpaI and BamHI	5	2	1	3
13

Table1

Diagnostic Gel:

Expected (if had LacZ Cassette)

LowSenBack.PNG

miRNA_Repressor_Group_Gel.jpg

Conclusion: All the sensors have Jeremy's backbone and do not contain the LacZ cassette. Yay!

Jeremy miRNA Ultramers

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Elizabeth Strand

Dates: 2016-08-04 to 2016-08-05

Thursday, 8/4

Inventory

All in ~50 nM stock in 5 uL

50 nM in 5 ul is about 4.42 ng/uL

will make a ~2 ng/uL dilution of all of them in 2 uL total. So there is 4 uL @ ~50 nM left if we need it.

	A	B	C	D
1	Sensor	Plate	Fwd Location	Rev Location
2	hsa-miR-18a-3p	plate 3	K6	K6
3	hsa-miR-20a-3p	plate 3	I8	I8
4	hsa-miR-135a-3p	plate 3	I4	I4
5	hsa-miR-451a	plate 2	A17	B17
6	hsa-miR-34c-5p	plate 2	I15	J15
7	hsa-miR-9-5p	plate 2	I22	J22

Table1

PCR Amplification

PCR to make more ultramers from Jeremy Gam

For all ultramer #'s

	A	B	C	D
1	Nickname	Template	Forward Primer	Reverse Primer
2	#	# ultramer	iGEM 41 miR-jg-fwd	iGEM 42 miR-jg-rev

Table20

Prepare on Ice in this order

	A	B
1		Volume per rxn
2	Ultrapure H2O	18
3	Template (2 ng/uL)	2
4	Fwd Primer (10 uM)	2.5
5	Rev Primer (10 uM)	2.5
6	Q5 Master Mix (2X)	25
7	Total	32
8

Table2

Name of protocol: Q5 repressors B

	A	B	C	D
1	Step	Temperature (C)	Time (s)	Sample
2	Initial Denaturation	98	30
3	Cycle 30 x
4	Denature	98	10
5	Anneal	58	30	All
6	Extend	72	10
7	Cycle Ends
8	Final Extension	72	120
9	Store	4	forever

Table21

Friday, 8/5

Gel Extraction

	A	B	C
1	Lane	Nickname	Expected Length
2	1	Ladder
3	2	hsa-miR-18a-3p	136
4	3	hsa-miR-18a-3p	136
5	4	hsa-miR-20a-3p	136
6	5	hsa-miR-20a-3p	136
7	6	hsa-miR-135a-3p	136
8	7	hsa-miR-135a-3p	136
9	8	hsa-miR-451a	136
10	9	hsa-miR-451a	136
11	10	hsa-miR-34c-5p	136
12	11	hsa-miR-34c-5p	136
13	12	hsa-miR-9-5p	136
14	13	hsa-miR-9-5p	136
15	14	Ladder	136

Table22

8-8-16 ultramers1.PNG

Couldn't see ladder in lane 2 at all, ladder in lane 14 was super faint. I'm guessing things dissolved into TAE when I moved the loaded gel to the gel box station :( but I will do gel extraction anyways

Consolidated multiple bands of one miRNA to same tube.

	A	B	C	D	E
1	Tube	Weight (mg)	QG Added (uL)	Isopropanol Added (uL)	Nanodrop (ng/uL)
2	hsa-miR-18a-3p	242	1452	242	3.1
3	hsa-miR-20a-3p	267	1602	267	9.9
4	hsa-miR-135a-3p	213	1278	213	7.6
5	hsa-miR-451a	199	1194	199	17.2
6	hsa-miR-34c-5p	272	1632	272	9.9
7	hsa-miR-9-5p	237	1422	237	23.1

Table23

Stored in our glycerol stock box in -20C

	A	B	C	D
1	Step	Temperature (C)	Time (s)	Sample
2	Initial Denaturation	98	30
3	Cycle 30 x
4	Denature	98	10
5	Anneal	58	15	All
6	Extend	72	10
7	Cycle Ends
8	Final Extension	72	120
9	Store	4	forever

Table3

siRNA Inventory

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Wangui Mbuguiro

Date: 2016-08-12

Friday, 8/12

Resuspended in Water

	A	B	C	D	E
1		nmol	MW (g/mol)	Want (uM, In -80)	Water to add (uL)
2	siRNA-451a	122.6	13592.4	100	1226
3	siRNA-9-3p	122.1	13601.4	100	1221

Table1

Tandem Experiment

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Elizabeth Strand

Date: 2016-10-09

Sunday, 10/9

Plasmids

**could hypothetically use ERE5 instead, depends on what plasmids are available

ERE6:TP901_451a

ERE6:mKate

hef1a:eyfp_flippped

hef1a:eyfp

hef1a:BFP

alexa fluorphore?

Estrogen: 1 concentration -- 50 nM

siRNA-451a: 0nm, .5, 1, 5, 10

Wells:

single control X3

three color control

experimental wells X5

no estrogen control X5

untransfected