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From 2010.igem.org

Contents 1 Andreas 1.1 Assembly of SOD/yCCS⋅His constructs into pSB1K3 1.1.1 Plasmid prep 1.1.1.1 Sequencing 1.2 Enzyme inactivation 1.3 Transfer of RFP coding device to pEX 1.3.1 Colony restreaks 1.3.2 Gel verification of Dig pEX X+P and Dig pC.RFP X+P 1.3.2.1 Results 1.3.3 Digestion of new pEX vector 1.3.3.1 Gel verification 1.3.4 Ligation 1.3.5 Transformation 1.4 ON cultures 1.5 Assembly of His⋅SOD/yCCS constructs 1.5.1 Ligation 1.5.2 Transformation

Andreas

Assembly of SOD/yCCS⋅His constructs into pSB1K3

Continued from 21/8

Plasmid prep

†Samples concentrated in vacuum evaporator

DNA concentrations
	Before sample conc.		After sample conc.
Sample	Conc. [ng/μl]	A260/A280	Conc. [ng/μl]	A260/A280
pSB1K3.SOD⋅His 1†	61.50	1.86	100.9	1.80
pSB1K3.SOD⋅His 2†	76.45	1.78	126.1	1.83
pSB1K3.yCCS⋅His 2	171.5	1.88	–	–
pSB1K3.yCSS⋅His 3†	84.15	1.88	133.6	1.84

From 21/8 cultures

• E.Z.N.A Plasmid Mini kit I
• 50 μl elution volume

Sequencing

Samples prepared for sequencing:

• 15 μl DNA (>100 ng/μl)
• 1.5 μl 10 μM pSB-VR primer

Sample	Label	Seq #
pSB1K3.SOD⋅His 1	pK-SH1	938
pSB1K3.SOD⋅His 2	pK-SH2	939
pSB1K3.yCCS⋅His 2	pK-yH2	940
pSB1K3.yCCS⋅His 3	pK-yH3	941

Enzyme inactivation

Inactivated restriction enzymes in digestion samples from 19/8 and 20/8 in 80 °C, 10 min.

• Dig pEX X+P
• Dig pC.RFP X+P
• Dig His E+A
• Dig m-yCCS N+P
• Dig m-SOD N+P

Transfer of RFP coding device to pEX

Colony restreaks

Results from 21/8

All clones grew well on the Amp plate, but not on Km, indicating RFP has indeed been transfered from pSB1K3 to a target AmpR vector, and that pSB1K3 does not express AmpR.

I later realized that the transfer of RFP was not from pSB1K3, but from pSB1C3, making this restreak pointless.

Gel verification of Dig pEX X+P and Dig pC.RFP X+P

Ran a gel of the digestion samples from 19/8 to verify the sizes of vectors and inserts.

1 % agarose, 100 V, 1 h 20 min

Expected bands

• Dig pEX X+P
  • 4453 bp (vector)
  • 1237 bp (insert)
• Dig pC.RFP X+P
  • 2054 bp (pSB1C3 vector)
  • 1095 bp (RFP insert)

Results

The bands for "pC.RFP X+P" correspond well to what was expected. However, none of the "pEX X+P" bands do. It might be possible that this is actually a pMA vector, carrying a ≈800 bp insert (unclear what). This would explain why ligations and transformations from 19/8 resulted in red colonies, but colony PCR amplification with pEX primers doesn't result in any bands.

Digestion of new pEX vector

Used pEX vector prepared and verified 21/8 for new pEX digestion with XbaI and PstI.

[pEX] = 55.52 ng/μl

Digestion mix
10X FD buffer	3 μl	[DNA] = 33.3 ng/μl Inc.: 37 °C, 30 min
dH2O	7 μl
1 μg DNA (pEX)	18 μl
FD XbaI	1 μl
FD PstI	1 μl
	30 μl

Enzyme inactivation (

after

ligation): 80 °C, 5 min

Gel verification

1 % agarose, 100 V, 35 min

Results
pEX confirmed - bands correspond well.

Ligation

Ligation mix
100 ng vector (pEX)	3 μl	1/3 ratio	[Dig pEX X+P] = 33.3 ng/μl (23/8) [Dig pC.RFP X+P]=66.6 ng/μl (19/8) Inc.: 22 °C, 10 min
165 ng insert (RFP)	2.5 μl
5X Rapid Ligation buf.	4 μl
dH2O	9.5 μl
T4 DNA Ligase	1 μl
	20 μl

Transformation

Procedures based on quick transformation protocol:

• 1.5 μl ligation mix. 15 min incubation on ice.
• 30 s heat-shock in 42 °C

Plating onto 100 Amp LB agar. 37 °C ON.

ON cultures

Set ON cultures for preparation of glycerol stocks:

• 3 ml LB + Km 50:
  • pSB1K3.SOD⋅His 1
  • pSB1K3.SOD⋅His 2
  • pSB1K3.yCCS⋅His 2
  • pSB1K3.yCCS⋅His 3
• 3 ml LB + Amp 100:
  • pMA.His (Picked 19/8)
• pEX (Picked 19/8)

Inc. 30 °C, ON.

Assembly of His⋅SOD/yCCS constructs

Ligation

Used digestion samples from 20/8 for ligations. Ligation protocol identical to that from 20/8.

Transformation

Standard transformation protocol.

• 1 μl ligation mix
• Plating on Km 50.