Team:Wageningen UR/Journal/week12

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== Office work ==
== Office work ==
 +
'''PLRV'''
 +
 +
This week we designed and ordered all the primers we needed.
 +
[[File:primers.jpg]]
== Lab work ==
== Lab work ==
-
Biobrick BBa_J04450 was cloned and miniprepped.
+
'''General'''
-
''' Hepatitis B '''
+
BBa_J04450 (RFP coding device) transformation, digestion check
 +
*BBa_J04450 was solubilized from the Standard registry parts plate I with 10µl MQ water
 +
*Transformation of electrocompetent ''E.coli'' with the plasmid
 +
*Plated on LB-agar with corresponding antibiotic (chloramphenicol)
 +
*6 red-fluorescing colony were picked after overnight growth in 37°C
 +
*Subsequent miniprep
 +
 +
{| class="wikitable" style="text-align: center"
 +
|- style="font-style: italic"
 +
|sample
 +
|concentration (ng/µl)
 +
|260/280
 +
|-
 +
|A1
 +
|115.5
 +
|1.92
 +
|-
 +
|C1
 +
|96.0
 +
|1.98
 +
|-
 +
|C3
 +
|100.1
 +
|1.92
 +
|}
-
'''Tuesday:'''
+
*Digestion check with EcoRI and SpeI
-
Digestion of
+
[[File:RFP pSB1C3. digestion check.jpg|300px|center|thumb|''Figure 1: digestion BBa_J04450'']]
-
* Hepatitis B PCR product (with pre- and suffix) with Spe1 and Pst1
+
-
* BBa_J04500 (an IPTG inducible promoter with RBS) with Xba1 and Pst1
+
-
* BBa_PSB1K3.ml (linearized plasmid backbone) with EcoR1 and Pst1
+
 +
Digestion of BBa_K1970- 22/38
-
PCR purification on the digest as a replacement for the heat inactivation
+
From the transformation plate of week 9 a new colony was grown and subsequently a miniprep was done. The yield was about 160 ng/µl. To check if the transformed E.coli contains the right plasmid a digestion check with the restriction enzymes BamHI and BgEII was done.
 +
[[File:Digest BBa K197022-38.jpg|500px|center|thumb|''Figure 2: digestion BBa_K197022/38'']]
-
'''Wednesday:'''
+
''' CCMV '''
-
Ligation of
+
''18th July'' (Hugo)
-
* Hepatitis B PCR product with BBa_J04500
+
<br/>
-
* the Hepatitis B PCR product with BBa_PSB1K3.ml
+
First PCR on CCMV-Delta26 to create the CCMV_NEG construct.
-
''The ligations where done in duplo – once following the standard iGEM protocol and once using equimolar amounts of Hep B and the vector (DNA concentrations where measured by using NanoDrop)''
+
 +
Phusion Mastermix for 4 + 1 = 5 samples:
-
Electrotransformation of the 2 different constructs with DH5α
 
-
* Grow transformed E.coli on plates containing kanamycin
 
-
* A plasmid with a GFP coding device was used as a positive control (growing on a plate containing ampicillin)
 
-
 
-
'''Thursday:'''  
+
{| class="wikitable" style="text-align: center"
 +
|- style="font-style: italic"
 +
|Substance
 +
|Amount (µl)
 +
|-
 +
|H2O
 +
|176.25 uL
 +
|-
 +
|5x Phusion Buffer
 +
|50 uL
 +
|-
 +
|dNTP's
 +
|5 uL
 +
|-
 +
|Phusion enzyme
 +
|1.25 uL
 +
|-
 +
|Total
 +
|232.5 uL
 +
|}
 +
 
 +
Preparation samples:
 +
 
 +
 
 +
{| class="wikitable" style="text-align: center"
 +
|- style="font-style: italic"
 +
|Substance
 +
|Amount (µl)
 +
|-
 +
|Mastermix
 +
|44 uL
 +
|-
 +
|DNA template
 +
|1 uL
 +
|-
 +
|Forward Primer (10x diluted)
 +
|2.5 uL
 +
|-
 +
|Reverse Primer (10x diluted)
 +
|2.5 uL
 +
|-
 +
|Total
 +
|50 uL
 +
|}
 +
 
 +
PCR reaction:
 +
 
 +
{| class="wikitable" style="text-align: center"
 +
|- style="font-style: italic"
 +
|Step
 +
|Time
 +
|Temperature (°C)
 +
|-
 +
|Step 1
 +
|10 minutes
 +
|98 °C
 +
|-
 +
|Step 2
 +
|30 seconds
 +
|98 °C
 +
|-
 +
|Step 3
 +
|30 seconds
 +
|58 °C
 +
|-
 +
|Step 4
 +
|30 seconds
 +
|72 °C 
 +
|Return to step 2 (35 times)
 +
|-
 +
|Step 5
 +
|10 minutes
 +
|72 °C
 +
|-
 +
|Step 6
 +
|As long as it takes
 +
|4 °C
 +
|-
 +
|}
 +
 
 +
Gel:
 +
 
 +
{| class="wikitable" style="text-align: center"
 +
|- style="font-style: italic"
 +
|Lane
 +
|Sample
 +
|-
 +
|Lane 1
 +
|Marker
 +
|-
 +
|Lane 2
 +
|Negative control: No template, same primers as duplo’s
 +
|-
 +
|Lane 3
 +
|Positive control: Delta26-Histag CCMV with Forward + Reverse (suffix) primer.
 +
|-
 +
|Lane 4
 +
|duplo 1: CCMV-Delta26 with Forward (Part 2) + Reverse (suffix) primer.
 +
|-
 +
|Lane 5
 +
|duplo 2: CCMV-Delta26 with Forward (Part 2) + Reverse (suffix) primer.
 +
|}
 +
 
 +
[[File:Gel_18-07.tif|600px|center|thumb|''Figure 3: 1% agarose gel''</p>]]
 +
 
 +
''19th July'' (Hugo)
 +
<br/>
 +
Second PCR on CCMV-Delta26. Before PCR was started, fragments of the day before were purified using a PCR purification kit.
 +
<br/>
 +
<br/>
 +
''20th July'' (Hugo)
 +
<br/>
 +
PCR repeat of 18-07.
 +
<br/>
 +
<br/>
 +
 
 +
 
 +
''' HepB '''
 +
 
 +
* Digestion of the HepB core protein PCR product with pre- and suffix (from 11.July) and ligation into BBa_J04500 (an IPTG inducible promoter with RBS)as well as BBa_PSB1K3.ml (linearized plasmid backbone)
 +
* Electro transformation of these constructs with DH5α
 +
* Colony PCR (20 samples of the transformants containing HepB core protein + BBa_J04500; 10 samples of the transformants containing HepB core protein + BBa_PSB1K3.ml
 +
-> The ligation and transformation of the HepB core protein into the linear backbone (BBa_PSB1K3.ml) was not successful
 +
-> The colony PCR shows that we have 6 out of 20 colonies with the expected insert size (around 600bp)
 +
 
 +
 
 +
[[File:ColonyPCR_20July.png|200px|center|thumb|<p align="justify">''Figure 4: Colony PCR 20 July - HepB in BBa_J04500'' </p>]]
 +
 
-
Colony PCR
 
-
* of 20 colonies transformed with the Hep B + BBa_J04500 construct
 
-
* of 10 colonies transformed with the Hep B + BBa_PSB1K3.ml construct
 
-
''out of 20 samples taken from the transformation with the HepB + BBa_J04500 construct – 7 turned out to have the right insert. The colonies transformed with the Hep B + BBa_PSB1K3.ml construct did not have any Hep B inserts''
 
'''TuYV'''
'''TuYV'''
-
* Mach1 electrocompetent ''E. coli'' cells were transformed with last week's ligation mixtures. Results were unsatisfying.
+
* Mach1 electrocompetent ''E. coli'' cells were transformed with last week's ligation mixtures (TuYV coat protein and TuYV coat protein with his-tag). Results were unsatisfying. No colonies were showed on the sample plates.  
* 'Coat Protein + readthrough' gene amplicons were digested, ligated into pSB1K3 backbone and transformed into our electrocompetent Mach1 ''E. coli'' cells. After two attempts and a colony PCR, results were still unsatisfying.
* 'Coat Protein + readthrough' gene amplicons were digested, ligated into pSB1K3 backbone and transformed into our electrocompetent Mach1 ''E. coli'' cells. After two attempts and a colony PCR, results were still unsatisfying.
Line 59: Line 205:
After a two week quest for PLRV infected plant material, we obtained infected potato leafs from the Dutch General Inspection Service for Agricultural seed and seed potatoes.  
After a two week quest for PLRV infected plant material, we obtained infected potato leafs from the Dutch General Inspection Service for Agricultural seed and seed potatoes.  
-
The figure below shows the purity of isolated RNA. We isolated RNA of different potato cultivars.
+
----
 +
 
 +
'''DLS boundary experiment'''
-
[[File:rna2.jpg]]
 
 +
''19th July'' (Mark)
 +
<ul>
 +
<li>DLS measurment: 20 runs, 45 seconds</li>
 +
<ul>
 +
<li>Changed 4 times to different vials to see if the vials are important to the measurement</li>
 +
</ul>
 +
</ul>
----
----
[[https://2012.igem.org/Team:Wageningen_UR/Journal/week11 previous week]]          [[https://2012.igem.org/Team:Wageningen_UR/Journal/week13 next week]]
[[https://2012.igem.org/Team:Wageningen_UR/Journal/week11 previous week]]          [[https://2012.igem.org/Team:Wageningen_UR/Journal/week13 next week]]

Latest revision as of 03:25, 27 September 2012

week 12: 16 july - 22 july

Office work

PLRV

This week we designed and ordered all the primers we needed.

Primers.jpg

Lab work

General

BBa_J04450 (RFP coding device) transformation, digestion check

  • BBa_J04450 was solubilized from the Standard registry parts plate I with 10µl MQ water
  • Transformation of electrocompetent E.coli with the plasmid
  • Plated on LB-agar with corresponding antibiotic (chloramphenicol)
  • 6 red-fluorescing colony were picked after overnight growth in 37°C
  • Subsequent miniprep
sample concentration (ng/µl) 260/280
A1 115.5 1.92
C1 96.0 1.98
C3 100.1 1.92
  • Digestion check with EcoRI and SpeI
Figure 1: digestion BBa_J04450

Digestion of BBa_K1970- 22/38

From the transformation plate of week 9 a new colony was grown and subsequently a miniprep was done. The yield was about 160 ng/µl. To check if the transformed E.coli contains the right plasmid a digestion check with the restriction enzymes BamHI and BgEII was done.

Figure 2: digestion BBa_K197022/38

CCMV

18th July (Hugo)
First PCR on CCMV-Delta26 to create the CCMV_NEG construct.

Phusion Mastermix for 4 + 1 = 5 samples:


Substance Amount (µl)
H2O 176.25 uL
5x Phusion Buffer 50 uL
dNTP's 5 uL
Phusion enzyme 1.25 uL
Total 232.5 uL

Preparation samples:


Substance Amount (µl)
Mastermix 44 uL
DNA template 1 uL
Forward Primer (10x diluted) 2.5 uL
Reverse Primer (10x diluted) 2.5 uL
Total 50 uL

PCR reaction:

Step Time Temperature (°C)
Step 1 10 minutes 98 °C
Step 2 30 seconds 98 °C
Step 3 30 seconds 58 °C
Step 4 30 seconds 72 °C Return to step 2 (35 times)
Step 5 10 minutes 72 °C
Step 6 As long as it takes 4 °C

Gel:

Lane Sample
Lane 1 Marker
Lane 2 Negative control: No template, same primers as duplo’s
Lane 3 Positive control: Delta26-Histag CCMV with Forward + Reverse (suffix) primer.
Lane 4 duplo 1: CCMV-Delta26 with Forward (Part 2) + Reverse (suffix) primer.
Lane 5 duplo 2: CCMV-Delta26 with Forward (Part 2) + Reverse (suffix) primer.

File:Gel 18-07.tif

19th July (Hugo)
Second PCR on CCMV-Delta26. Before PCR was started, fragments of the day before were purified using a PCR purification kit.

20th July (Hugo)
PCR repeat of 18-07.


HepB

  • Digestion of the HepB core protein PCR product with pre- and suffix (from 11.July) and ligation into BBa_J04500 (an IPTG inducible promoter with RBS)as well as BBa_PSB1K3.ml (linearized plasmid backbone)
  • Electro transformation of these constructs with DH5α
  • Colony PCR (20 samples of the transformants containing HepB core protein + BBa_J04500; 10 samples of the transformants containing HepB core protein + BBa_PSB1K3.ml

-> The ligation and transformation of the HepB core protein into the linear backbone (BBa_PSB1K3.ml) was not successful -> The colony PCR shows that we have 6 out of 20 colonies with the expected insert size (around 600bp)


Figure 4: Colony PCR 20 July - HepB in BBa_J04500



TuYV

  • Mach1 electrocompetent E. coli cells were transformed with last week's ligation mixtures (TuYV coat protein and TuYV coat protein with his-tag). Results were unsatisfying. No colonies were showed on the sample plates.
  • 'Coat Protein + readthrough' gene amplicons were digested, ligated into pSB1K3 backbone and transformed into our electrocompetent Mach1 E. coli cells. After two attempts and a colony PCR, results were still unsatisfying.


PLRV

After a two week quest for PLRV infected plant material, we obtained infected potato leafs from the Dutch General Inspection Service for Agricultural seed and seed potatoes.

DLS boundary experiment


19th July (Mark)

  • DLS measurment: 20 runs, 45 seconds
    • Changed 4 times to different vials to see if the vials are important to the measurement


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