Team:Stockholm/24 September 2010

From 2010.igem.org

Revision as of 15:54, 26 October 2010 by NinaSchiller (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

Andreas

Plasmid prep

From 23/9 ON cultures

Omega E.Z.N.A. Plasmid Miniprep kit I.

New plasmid prep buffers

† = *unsure value due to bad blank sample.*
DNA concentration
Sample	Conc [ng/μl]	A₂₆₀/A₂₈₀
pSB1A2.RBS.yCCS 3	195.5	1.81
pSB1A2.RBS.yCCS 4	165.7	1.85
pEX.N-TAT⋅SOD⋅His 3†	60.00	1.83
pEX.N-TAT⋅SOD⋅His 4†	90.00	1.73
pSB1K3.N-TAT⋅SOD⋅His 4	130.5	1.84
pSB1K3.N-TAT⋅SOD⋅His 5†	60.00	1.88
pSB1K3.N-Tra10⋅SOD⋅His 5†	90.00	1.93
pSB1K3.N-LMWP⋅SOD⋅His 1	258.2	1.87
pSB1C3.N-LMWP⋅SOD⋅His 1	155.4	1.82
pSB1C3.N-LMWP⋅SOD⋅His 4	331.9	1.80

Cloning and assembly

Digestions

[pEX.RFP] = 44.0 ng/μl

	pA.RBS. yCCS (3) X+P	pEX.N-TAT. SH (4) X+P	pK.N-TAT. SH (4) S+P	pK.N-Tra10. SH (5) X+P	pK.N-Tra10. SH (5) S+P	pC.N-LMWP. SH (4) X+P	pC.N-LMWP. SH (4) S+P	pEX.RFP X+P
10X FastDigest buffer	2	2	2	2	2	2	2	3
DNA (1 μg)	5	12.5	8	12.5	12.5	4	4	22
dH₂O	11	3.5	7	3.5	3.5	12	12	3
FD XbaI	1	1	0	1	0	1	0	1
FD PstI	1	1	1	1	1	1	1	1
FD SpeI	0	0	1	0	1	0	1	0
	20 μl	20 μl	20 μl	20 μl	20 μl	20 μl	20 μl	30 μl

Incubation: 37 °C, 1 h

Gel verification

Gel verification of sample digestions.
4 μl λ; 3 μl sample.
1 kb λ = O'GeneRuler 1 kb DNA ladder. 50 bp λ = GeneRuler 50 bp DNA ladder.

Due to the risk of pSB1A2.RBS.yCCS and pEX.N-TAT⋅SOD⋅His having previously been mixed up, a gel was run on the digested samples (after 15 min incubation) to verify the excised insert size. Remaining samples were also run on the gel to verify digestion.

1 % agarose, 120 V

Expected bands

pSB1A2.RBS.yCCS X+P: 806 bp, 2061 bp
pEX.N-TAT⋅SOD⋅His X+P: 558 bp, 4453 bp
pSB1K3.N-TAT⋅SOD⋅His S+P: ≈2730 bp
pSB1K3.N-Tra10⋅SOD⋅His X+P: 588 bp, 2188 bp
pSB1K3.N-Tra10⋅SOD⋅His S+P: ≈2760 bp
pSB1C3.N-LMWP⋅SOD⋅His X+P: 567 bp, 2054 bp
pSB1C3.N-LMWP⋅SOD⋅His S+P: ≈2610 bp
pEX.RFP X+P: 1095 bp, 4453 bp

Results
Seemingly correct bands for samples 1, 3, 4, 5 and 8. More unsure results for 6 and 7, while 2 seems to contain more than one insert, or has been digested at several places.

Nina

Overnight culture

I inoculated protein A#5_TAT, _LMWP & _Tra10 all from colony #1 on each dish.

12 ml LB + 24 ul chloramphenicol.

Concentration measurement

Protein A#5_CPP_TAT_C cons: 95ng/ul 95/10 = 9.5ng/ul λ260 0.019 λ280 0.009 λ315 -0.002

Vector with CPP_TAT_C conc: 95 ng/ul

95ng/ul * Volume = 25ng/ul * 5 ul

Volume = 1.3 ul sample and fill up to 5 ml with H2O.

IgG N+P cons: 30ng/ul 30/2.5 = 12ng/ul λ260 0.007 λ280 0.006 λ315 0.000

IgG A+E cons: 40ng/ul 40/2.5 = 16ng/ul λ260 0.007 λ280 0.007 λ315 0.001

Ligation

Vector CPP_TAT_C 1 ul
Protein A gene 21 ul
Quick ligase 1 ul
Quicke ligase buffer 2X 23 ul

Vector LMWP, TAT & Tra10 0.5 ul each
gene IgG (N+P) 7.5 ul each
Quick ligation 1 ul
Quick ligation buffer 2X 9 ul each

Vector CPP_TAT_C 1 ul
gene IgG (A+E) 17 ul
Quick ligation 1 ul
Quick ligation buffer 2X 19 ul

Transformation

I transformed the ligations with 10 ul in 100 ul Top 10 cloning cells.

Digestion

H2O 15 ul
Fastdigest buffer 10X 2 ul
DNA 2 ul
Restriction enzyme NgoMIV 1 ul
Restriction enzyme PstI 1 ul (Add after 1.5 h incubation in 37 °C and incubate in 30 min)

Inactivate in 80 °C for 30 min.

Added CIAP 1 ul and incubate 1 h in 37 °C.