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Revision as of 11:35, 6 August 2013 (view source) FelicityHeath (Talk | contribs) (Created page with "== Liquid cultures from the 30/07/2013 == {| class="wikitable" |- ! Liquid culture !! Did it work? |- | K592018 || yes |- | B0015 || yes |- | K592022 || no |- | K864404 || no |-...") ← Older edit		Revision as of 12:20, 6 August 2013 (view source) FelicityHeath (Talk | contribs) Newer edit →
Line 43:		Line 43:
	3 - negative control (water)		3 - negative control (water)
-	4 -	+	4 - posiive control (RFP cut with E + S)
		+
		+	5 - positive control (RFP cut with X + P)
		+
		+
		+	These were then vortexed.
		+
		+
		+	Master mix
		+
		+	120ul nuclease free water
		+
		+	20ul 10x fast digest buffer w/green.
		+
		+	VORTEX.
		+
		+
		+	15ul into each PCR tube.
		+
		+	Add 5ul DNA
		+
		+	Vortex
		+
		+	Incubate at 37°C for 10 minutes.
		+
		+
		+	== Results of Nanodrop ==
		+
		+
		+	{| class="wikitable"
		+	|-
		+	! Culture !! Nanodrop concentration (ng/ul) !! Digest (ul)
		+	|-
		+	| K592018 || 130.4 || 1.92
		+	|-
		+	| B0015 || 81.6 || 3.06
		+	|-
		+	| S05058 || 111.0 || 2.25
		+	|-
		+	| RFP || 104.4
		+	|}
		+
		+
		+	== Restriction Digest - Victoria's Recipe ==
		+
		+	We are attaching terminators (B0015) to K592018 (RBS+Cph8) and S05058 (RBS+cyan). We are putting them in the AMP plasmid.
		+
		+
		+	K592018 - cut with E + X (part A)
		+
		+	S05058 - cut with E + X (part A)
		+
		+	B0015 - cut with E + S (part B)
		+
		+
		+	== Part A ==
		+
		+	250/x = ul DNA needed.            x = Nanodrop value
		+
		+
		+	250ng DNA
		+
		+	2.5ul NEB2 buffer (vortex)
		+
		+	0.5ul BSA (vortex)
		+
		+	0.5ul EcoR1
		+
		+	0.5ul xba1
		+
		+	make up to 20ul with no nuclease water.
		+
		+
		+	== Part B ==
		+
		+
		+	250ng DNA
		+
		+	2.5ul NEB2 (vortex)
		+
		+	0.5ul BSA (vortex)
		+
		+	0.5ul EcoR1
		+
		+	0.5ul Spel
		+
		+	make up to 20ul with no-nuclease water.
		+
		+
		+	==Vector ==
		+
		+
		+	250ng plasmid (comes as 25ng/ul)
		+
		+	2.5ul NEB2 (vortex)
		+
		+	0.5ul BSA (vortex)
		+
		+	0.5ul EcoR1
		+
		+	0.5ul Pstl
		+
		+	0.5ul Dnpl
		+
		+	make up to 20ul with no-nuclease water.
		+
		+
		+	== Positive controls (E + S) ==
		+
		+
		+	250ng DNA
		+
		+	3.0ul NEB2 (vortex)
		+
		+	0.5ul EcoR1
		+
		+	0.5ul Spel
		+
		+	make up to 20ul
		+
		+
		+	== Positive control (X + P) ==
		+
		+
		+	250ng DNA
		+
		+	3.0ul NEB2 (vortex)
		+
		+	0.5ul xba1
		+
		+	0.5ul Pstl
		+
		+	make up to 20ul.
		+
		+
		+	== Negative control ==
		+
		+
		+	No DNA
		+
		+	0.5ul EcoR1
		+
		+	0.5ul Spe1
		+
		+	3.0ul NEB2 - vortex
		+
		+	make up to 20ul.
		+
		+
		+	Thermocycler:
		+
		+	37°C for 30 minutes
		+
		+	80°C for 20 minutes
		+
		+	4°C hold
		+
		+
		+	Add water and DNA first, then enzymes, buffer and BSA.
		+
		+	Make sure NEB and BSA are completely defrosted and vortex before using.
		+
		+	Vortex and spin down before using the thermocycler.
		+
		+
		+	- Had to do a sure clean on the RFP.
		+
		+	80ul of RFP, original Nano drop was 12.6ng/ul.

---

Revision as of 12:20, 6 August 2013

Contents 1 Liquid cultures from the 30/07/2013 2 Mini Prep 3 Digest 4 Results of Nanodrop 5 Restriction Digest - Victoria's Recipe 6 Part A 7 Part B 8 Vector 9 Positive controls (E + S) 10 Positive control (X + P) 11 Negative control

Liquid cultures from the 30/07/2013

Liquid culture	Did it work?
K592018	yes
B0015	yes
K592022	no
K864404	no
K592011	no
S05058	yes

We then mini-prepped the liquid cultures that worked.

Mini Prep

We centrifuged the entire falcon tube for 10 minutes at 5000 rpm.

Then resuspended the whole pellet.

Nuclease free water was then run through instead of elution buffer, this was done twice.

Digest

Half was run on a gel, half was kept aside for ligation.

1 - RBS + Cph8 (cut with E + S)

2 - B0015 (cut with X + P)

3 - negative control (water)

4 - posiive control (RFP cut with E + S)

5 - positive control (RFP cut with X + P)

These were then vortexed.

Master mix

120ul nuclease free water

20ul 10x fast digest buffer w/green.

VORTEX.

15ul into each PCR tube.

Add 5ul DNA

Vortex

Incubate at 37°C for 10 minutes.

Results of Nanodrop

Culture	Nanodrop concentration (ng/ul)	Digest (ul)
K592018	130.4	1.92
B0015	81.6	3.06
S05058	111.0	2.25
RFP	104.4

Restriction Digest - Victoria's Recipe

We are attaching terminators (B0015) to K592018 (RBS+Cph8) and S05058 (RBS+cyan). We are putting them in the AMP plasmid.

K592018 - cut with E + X (part A)

S05058 - cut with E + X (part A)

B0015 - cut with E + S (part B)

Part A

250/x = ul DNA needed. x = Nanodrop value

250ng DNA

2.5ul NEB2 buffer (vortex)

0.5ul BSA (vortex)

0.5ul EcoR1

0.5ul xba1

make up to 20ul with no nuclease water.

Part B

250ng DNA

2.5ul NEB2 (vortex)

0.5ul BSA (vortex)

0.5ul EcoR1

0.5ul Spel

make up to 20ul with no-nuclease water.

Vector

250ng plasmid (comes as 25ng/ul)

2.5ul NEB2 (vortex)

0.5ul BSA (vortex)

0.5ul EcoR1

0.5ul Pstl

0.5ul Dnpl

make up to 20ul with no-nuclease water.

Positive controls (E + S)

250ng DNA

3.0ul NEB2 (vortex)

0.5ul EcoR1

0.5ul Spel

make up to 20ul

Positive control (X + P)

250ng DNA

3.0ul NEB2 (vortex)

0.5ul xba1

0.5ul Pstl

make up to 20ul.

Negative control

No DNA

0.5ul EcoR1

0.5ul Spe1

3.0ul NEB2 - vortex

make up to 20ul.

Thermocycler:

37°C for 30 minutes

80°C for 20 minutes

4°C hold

Add water and DNA first, then enzymes, buffer and BSA.

Make sure NEB and BSA are completely defrosted and vortex before using.

Vortex and spin down before using the thermocycler.

- Had to do a sure clean on the RFP.

80ul of RFP, original Nano drop was 12.6ng/ul.