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Revision as of 10:34, 24 August 2010 (view source) AndreasConstantinou (Talk | contribs) (→Gel verification) ← Older edit		Latest revision as of 02:01, 28 October 2010 (view source) JohanNordholm (Talk | contribs) m
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	{{Stockholm/Top2}}		{{Stockholm/Top2}}
		+
		+	==Nina==
		+
		+	===Sequencing of fusion protein===
		+
		+	I prepared one tube of the fusion protein made up by protein A and IgG protease:
		+
		+	15 ul plasmid from a mini-prep and 1.5 ul (10uM) primer VF2 of the bank vectors (C) verification primers.
		+
		+	*ASB0045 B00
		+
		+	===Ligation of protein A in peX===
		+
		+	I performed a ligation of protein A with the peX vector in order to induce the protein with IPTG o be able to see that the protein can become overexpressed in BL21 cells.
		+
		+	Ligation:
		+
		+	I wanted 25 ng vector
		+
		+	90 ng/ul / X = 25 ng/ul
		+
		+	X = 3 which means a 1:3 dilution. 1 ul vector sample and 2 ul dH2O. This results in 30 ng vector which is OK.
		+
		+	I wanted ~4 times more gene than vector. Usually it is enough with 3 times more gene than vector but since this gene is pretty small (180 bp) I prefer to have a 4X more gene.
		+
		+	4 * 30 = 120
		+
		+	120 ng/ul / 30ng/ul = 4 times more gene than vector.
		+
		+	*Vector peX: 1 ul
		+	*Gene protein A: 5 ul
		+	*Quick ligase: 1 ul
		+	*2X quick ligase buffer: 7 ul
		+
		+	Tot Volume: 14 ul
		+
		+	I incubated in RT for 15 min.
		+
		+	===PCR of ligation product===
		+
		+	I wanted to check if I had a correct ligation before I transformed it into BL21 cells. Therefore I performed a PCR on the ligation product with peX verification primers.
		+
		+	PCR mix:
		+
		+	*MgCl 1 ul
		+	*Buffer 5X 10 ul
		+	*dNTP 1 ul
		+	*primer peX forward 3 ul
		+	*primer peX reverse 3 ul
		+	*polymerase PjuX7 1 ul
		+	*H2O 30 ul
		+	*Template 1 ul
		+
		+	PCR prgm:
		+
		+	===Agarose gel on protein A===
		+
		+	I ran an agarose gel 1 % 100 V on the PCR product of protein A in the peX vector in order to verify that the ligation has occured properly.
		+
		+	Ladder: FastRuler™ Low Range DNA Ladder, ready-to-use, 50-1500 bp Fermentas
		+
		+	[[Image:Bild10.jpg|250px]]
		+
		+	The gel results show that I have had a good ligation since I got a band just below 400 on the ladder which I expected. The protein A gene is 180 nt and the verification primers add 200 nt which give a 380 nt band. This allows for a transformation tomorrow of the ligation product into BL21 cells.
		+
		+	----
	==Andreas==		==Andreas==
Line 199:		Line 265:
	*1 μl ligation mix		*1 μl ligation mix
	*Plating on Km 50.		*Plating on Km 50.
		+
		+	==Johan==
		+
		+	* PCR of bFGF from original vector
		+
		+	1 µl Pfu polymerase
		+
		+	5 µl 10x Pfu buffer
		+
		+	36 µl H2O
		+
		+	1 µl DNA
		+
		+	1 µl dNTP
		+
		+	3 µl bFGF for primer
		+
		+	3 µl bFGF rev primer
		+
		+	{{Stockholm/Footer}}

---

Latest revision as of 02:01, 28 October 2010

Contents 1 Nina 1.1 Sequencing of fusion protein 1.2 Ligation of protein A in peX 1.3 PCR of ligation product 1.4 Agarose gel on protein A 2 Andreas 2.1 Assembly of SOD/yCCS⋅His constructs into pSB1K3 2.1.1 Plasmid prep 2.1.1.1 Sequencing 2.2 Enzyme inactivation 2.3 Transfer of RFP coding device to pEX 2.3.1 Colony restreaks 2.3.2 Gel verification of Dig pEX X+P and Dig pC.RFP X+P 2.3.2.1 Results 2.3.3 Digestion of new pEX vector 2.3.3.1 Gel verification 2.3.4 Ligation 2.3.5 Transformation 2.4 ON cultures 2.5 Assembly of His⋅SOD/yCCS constructs 2.5.1 Ligation 2.5.2 Transformation 3 Johan

Nina

Sequencing of fusion protein

I prepared one tube of the fusion protein made up by protein A and IgG protease:

15 ul plasmid from a mini-prep and 1.5 ul (10uM) primer VF2 of the bank vectors (C) verification primers.

• ASB0045 B00

Ligation of protein A in peX

I performed a ligation of protein A with the peX vector in order to induce the protein with IPTG o be able to see that the protein can become overexpressed in BL21 cells.

Ligation:

I wanted 25 ng vector

90 ng/ul / X = 25 ng/ul

X = 3 which means a 1:3 dilution. 1 ul vector sample and 2 ul dH2O. This results in 30 ng vector which is OK.

I wanted ~4 times more gene than vector. Usually it is enough with 3 times more gene than vector but since this gene is pretty small (180 bp) I prefer to have a 4X more gene.

4 * 30 = 120

120 ng/ul / 30ng/ul = 4 times more gene than vector.

• Vector peX: 1 ul
• Gene protein A: 5 ul
• Quick ligase: 1 ul
• 2X quick ligase buffer: 7 ul

Tot Volume: 14 ul

I incubated in RT for 15 min.

PCR of ligation product

I wanted to check if I had a correct ligation before I transformed it into BL21 cells. Therefore I performed a PCR on the ligation product with peX verification primers.

PCR mix:

• MgCl 1 ul
• Buffer 5X 10 ul
• dNTP 1 ul
• primer peX forward 3 ul
• primer peX reverse 3 ul
• polymerase PjuX7 1 ul
• H2O 30 ul
• Template 1 ul

PCR prgm:

Agarose gel on protein A

I ran an agarose gel 1 % 100 V on the PCR product of protein A in the peX vector in order to verify that the ligation has occured properly.

Ladder: FastRuler™ Low Range DNA Ladder, ready-to-use, 50-1500 bp Fermentas

The gel results show that I have had a good ligation since I got a band just below 400 on the ladder which I expected. The protein A gene is 180 nt and the verification primers add 200 nt which give a 380 nt band. This allows for a transformation tomorrow of the ligation product into BL21 cells.

---

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.

Johan

• PCR of bFGF from original vector

1 µl Pfu polymerase

5 µl 10x Pfu buffer

36 µl H2O

1 µl DNA

1 µl dNTP

3 µl bFGF for primer

3 µl bFGF rev primer