Team:Stockholm/23 August 2010

From 2010.igem.org

(Difference between revisions)
(New page: {{Stockholm/Top2}} ==Andreas==)
m
 
(9 intermediate revisions not shown)
Line 1: Line 1:
{{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==
 +
 +
===Assembly of SOD/yCCS⋅His constructs into pSB1K3===
 +
''Continued from 21/8''
 +
====Plasmid prep====
 +
{|border="1" cellpadding="1" cellspacing="0" align="right"
 +
|+ align="bottom"|†Samples concentrated in vacuum evaporator
 +
!colspan="5"|DNA concentrations
 +
|-
 +
|width="130"| 
 +
|colspan="2" align="center"|Before sample conc.
 +
|colspan="2" align="center"|After sample conc.
 +
|-
 +
!Sample
 +
!width="90"|Conc. [ng/μl]
 +
!width="70"|A<sub>260</sub>/A<sub>280</sub>
 +
!width="90"|Conc. [ng/&mu;l]
 +
!width="70"|A<sub>260</sub>/A<sub>280</sub>
 +
|-
 +
|pSB1K3.SOD&sdot;His 1&dagger;
 +
|align="center"|61.50
 +
|align="center"|1.86
 +
|align="center"|100.9
 +
|align="center"|1.80
 +
|-
 +
|pSB1K3.SOD&sdot;His 2&dagger;
 +
|align="center"|76.45
 +
|align="center"|1.78
 +
|align="center"|126.1
 +
|align="center"|1.83
 +
|-
 +
|pSB1K3.yCCS&sdot;His 2
 +
|align="center"|171.5
 +
|align="center"|1.88
 +
|align="center"|&ndash;
 +
|align="center"|&ndash;
 +
|-
 +
|pSB1K3.yCSS&sdot;His 3&dagger;
 +
|align="center"|84.15
 +
|align="center"|1.88
 +
|align="center"|133.6
 +
|align="center"|1.84
 +
|-
 +
|}
 +
''From 21/8 cultures''
 +
*E.Z.N.A Plasmid Mini kit I
 +
*50 &mu;l elution volume
 +
 +
=====Sequencing=====
 +
Samples prepared for sequencing:
 +
*15 &mu;l DNA (>100 ng/&mu;l)
 +
*1.5 &mu;l 10 &mu;M pSB-VR primer
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!Sample
 +
!Label
 +
!Seq #
 +
|-
 +
|width="130"|pSB1K3.SOD&sdot;His 1
 +
|width="50"|pK-SH1
 +
|width="45" align="center"|938
 +
|-
 +
|pSB1K3.SOD&sdot;His 2
 +
|pK-SH2
 +
|align="center"|939
 +
|-
 +
|pSB1K3.yCCS&sdot;His 2
 +
|pK-yH2
 +
|align="center"|940
 +
|-
 +
|pSB1K3.yCCS&sdot;His 3
 +
|pK-yH3
 +
|align="center"|941
 +
|}
 +
 +
===Enzyme inactivation===
 +
Inactivated restriction enzymes in digestion samples from 19/8 and 20/8 in 80 &deg;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====
 +
[[image:Gelver_dig_pEX_pC.RFP_23aug.png|200px|thumb|right|'''Gel verification of digested pEX and pSB1C3.RFP samples from 19/8.'''<br />Loading vol.: 4 &mu;l &lambda;; 4 &mu;l sample.<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
 +
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'''<br />
 +
*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 &asymp;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.
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|+ align="bottom"|[pEX] = 55.52 ng/&mu;l
 +
!colspan="3"|Digestion mix
 +
|-
 +
|width="110"|'''10X FD buffer'''
 +
|width="30" align="center"|3 &mu;l
 +
|width="110" rowspan="6"|[DNA] = 33.3 ng/&mu;l<br /><br />Inc.: 37 &deg;C, 30 min
 +
|-
 +
|'''dH<sub>2</sub>O'''
 +
|align="center"|7 &mu;l
 +
|-
 +
|'''1 &mu;g DNA (pEX)'''
 +
|align="center"|18 &mu;l
 +
|-
 +
|'''FD XbaI'''
 +
|align="center"|1 &mu;l
 +
|-
 +
|'''FD PstI'''
 +
|align="center"|1 &mu;l
 +
|-
 +
|&nbsp;
 +
|align="center"|'''30 &mu;l'''
 +
|}
 +
 +
Enzyme inactivation (<ul>after</ul> ligation): 80 &deg;C, 5 min
 +
 +
=====Gel verification=====
 +
[[image:Gelver_dig_new-pEX_23aug.png|200px|thumb|right|'''Gel verification of new pEX vector digestion.'''<br />Loading vol.: 4 &mu;l &lambda;; 4 &mu;l sample.<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
 +
1 % agarose, 100 V, 35 min
 +
 +
'''Results'''<br />
 +
pEX confirmed - bands correspond well.
 +
 +
====Ligation====
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="4"|Ligation mix
 +
|-
 +
|'''100 ng vector (pEX)'''
 +
|align="center"|3 &mu;l
 +
|rowspan="2"|1/3<br />ratio
 +
|rowspan="6"|[Dig pEX X+P] = 33.3 ng/&mu;l (23/8)<br />[Dig pC.RFP X+P]=66.6 ng/&mu;l (19/8)<br /><br />Inc.: 22 &deg;C, 10 min
 +
|-
 +
|'''165 ng insert (RFP)'''
 +
|align="center"|2.5 &mu;l
 +
|-
 +
|'''5X Rapid Ligation buf.'''
 +
|align="center" colspan="2"|4 &mu;l
 +
|-
 +
|'''dH<sub>2</sub>O'''
 +
|align="center" colspan="2"|9.5 &mu;l
 +
|-
 +
|'''T4 DNA Ligase'''
 +
|align="center" colspan="2"|1 &mu;l
 +
|-
 +
|&nbsp;
 +
|align="center" colspan="2"|'''20 &mu;l'''
 +
|}
 +
 +
====Transformation====
 +
Procedures based on quick transformation protocol:
 +
*1.5 &mu;l ligation mix. 15 min incubation on ice.
 +
*30 s heat-shock in 42 &deg;C
 +
Plating onto 100 Amp LB agar. 37 &deg;C ON.
 +
 +
===ON cultures===
 +
Set ON cultures for preparation of glycerol stocks:
 +
*3 ml LB + Km 50:
 +
**pSB1K3.SOD&sdot;His 1
 +
**pSB1K3.SOD&sdot;His 2
 +
**pSB1K3.yCCS&sdot;His 2
 +
**pSB1K3.yCCS&sdot;His 3
 +
*3 ml LB + Amp 100:
 +
**pMA.His (Picked 19/8)
 +
*pEX (Picked 19/8)
 +
 +
Inc. 30 &deg;C, ON.
 +
 +
===Assembly of His&sdot;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 &mu;l 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
 +
 +
{{Stockholm/Footer}}

Latest revision as of 02:01, 28 October 2010

Home Project Idea Lab Work Results Modelling Team       Follow us on and        2010.igem.org

↳   Notebook Safety Protocols


Contents

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

Bild10.jpg

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

Gel verification of digested pEX and pSB1C3.RFP samples from 19/8.
Loading vol.: 4 μl λ; 4 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.

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
Gel verification of new pEX vector digestion.
Loading vol.: 4 μl λ; 4 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.

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





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/

Retrieved from "http://2010.igem.org/Team:Stockholm/23_August_2010"