Team:Aachen/Notebook/Protocols/Molecular biological methods

From 2014.igem.org

(Difference between revisions)
(PCR)
(PCR)
 
(15 intermediate revisions not shown)
Line 119: Line 119:
|| primers binding in the vector upstream and downstream of the insert or one in insert and one in vector are used
|| primers binding in the vector upstream and downstream of the insert or one in insert and one in vector are used
||  
||  
-
||
+
|| general reaction setup and procedure shown in tables below
-
* 12.5 µl GoTaq Master Mix
+
-
* 1 µl primer_F
+
-
* 1 µl primer_R
+
-
* pick colony with tip and suspend in PCR tube
+
-
* 9.5 µl ddH<sub>2</sub>O
+
|-
|-
| '''gradient PCR'''
| '''gradient PCR'''
|| Finding optimal conditions for our primers to bind and the PCRs in general
|| Finding optimal conditions for our primers to bind and the PCRs in general
|| Several PCRs batches are run within the same thermocycler, differing in annealing temperature
|| Several PCRs batches are run within the same thermocycler, differing in annealing temperature
-
|| [http://2014.igem.org/Team:Aachen/Notebook/Wetlab/August#6th in August 6th]
+
|| [http://2014.igem.org/Team:Aachen/Notebook/Wetlab/August#6th August 6th]
||
||
|-
|-
Line 141: Line 136:
|| side directed mutagenesis
|| side directed mutagenesis
|| primers containing the desired base pair exchange/deletion/insertion are designed and used  
|| primers containing the desired base pair exchange/deletion/insertion are designed and used  
-
|| [http://2014.igem.org/Team:Aachen/Notebook/Wetlab/April#23th in April 23th]
+
|| [http://2014.igem.org/Team:Aachen/Notebook/Wetlab/April#23th April 23th]
|| '''S'''plicing by '''O'''verlapping '''E'''xtension PCR
|| '''S'''plicing by '''O'''verlapping '''E'''xtension PCR
|-
|-
Line 147: Line 142:
|| side directed mutagenesis
|| side directed mutagenesis
|| general procedure by [http://www.chem.agilent.com/library/usermanuals/Public/200523.pdf  Agilent]
|| general procedure by [http://www.chem.agilent.com/library/usermanuals/Public/200523.pdf  Agilent]
-
|| [http://2014.igem.org/Team:Aachen/Notebook/Wetlab/August#5th in August 5th]
+
|| [http://2014.igem.org/Team:Aachen/Notebook/Wetlab/August#5th August 5th]
|| It was conducted by Vera at the laboratories of the Schwaneberg Group with supervision by Dr. rer. nat. Ljubica Vojcic.
|| It was conducted by Vera at the laboratories of the Schwaneberg Group with supervision by Dr. rer. nat. Ljubica Vojcic.
|}
|}
</center>
</center>
 +
 +
<div style="float:left;margin-right:1em;">
 +
{| class="wikitable"
 +
|+ general reaction procedure of a colony/check PCR
 +
! parameter !! duration !! temp [°C] !!
 +
|-
 +
| denature||5:00||95 ||
 +
|-
 +
| '''anneal'''||'''00:30'''||'''56''' || rowspan="3" | '''30 cycles'''
 +
|-
 +
| '''elongate'''||'''01:00 per kb'''||'''72'''
 +
|-
 +
| '''denature'''||'''00:30'''||'''95'''
 +
|-
 +
| elongate||05:00||72 || rowspan="2" |
 +
|-
 +
| store||forever||8
 +
|}
 +
</div>
 +
 +
<div style="float:left;margin-right:1em;">
 +
{| class="wikitable"
 +
|+ general reaction setup of a colony/check PCR
 +
! volume !! component
 +
|-
 +
| 12.5 µl||GoTaq Master Mix
 +
|-
 +
| 1 µl||primer_F
 +
|-
 +
| 1 µl||primer_R
 +
|-
 +
| 9.5 µl||ddH<sub>2</sub>O
 +
|-
 +
| ||pick colony with tip and suspend in PCR tube
 +
 +
|}
 +
</div>
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
<!--
<!--
=== Colony PCR /Check PCR ===
=== Colony PCR /Check PCR ===
''Does the cells contain the correct insert/plasmid and does the insert have the expected length?''
''Does the cells contain the correct insert/plasmid and does the insert have the expected length?''
-
'''With GoTaq Mast Mix'''
 
* 12.5 µl GoTaq Master Mix
* 12.5 µl GoTaq Master Mix
* 1 µl primer_F
* 1 µl primer_F

Latest revision as of 17:58, 17 October 2014

Molecular Biological Methods

We used different molecular biological methods in our project. All used methods are listed below.

Cloning

Plasmid Preparation

Plasmid preparation is a method for isolating plasmids from bacterial cell cultures. In this work the illustra™ plasmidPrep MIni Spin Kit (GE Healthcare) was used. After the cells are lysed, the lysate is applied to a mini column binding plasmid DNA to a silica membrane in the presence of chaotropic salts. Following a washing step, the DNA is eluted with bidest. water. Unless stated otherwise the plasmid preparation was performed following the manufacturer’s manual.

DNA-Purification

Some molecular biological methods require a purification of DNA after amplification or modification. In this work, the illustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare) was used. In the presence of chaotropic salts the nucleic acids are bound the glass fiber fleece in the Filter Tube while other substances are removed by the washing steps. Afterwards purified DNA fragments are be eluted with bidest. water. Unless stated otherwise the DNA purification was performed following the manufacturer’s manual.

Restriction Digest

Restriction endonucleases are used to cut double stranded DNA molecules at specific, usually palindromic base sequences. Unless stated otherwise the restriction digest was performed for 1 h at 37°C. To prevent religation of digested plasmids the DNA was dephosphorylated by addition of alkaline phosphatase and another incubation for 30 min at 37°C.

Ligation

Ligation in the context of molecular biology is the enzymatic joining of previously restricted nucleic acid fragments by synthesis of new bonds with simultaneous breakdown of ATP. A linearized plasmid functions as a vector for DNA inserts and all fragments are connected via ligation resulting in a new circular plasmid carrying the insert DNA. A typical reaction setup consists of a ligase, ligase buffer and the DNA fragments while the molar ratio of vector to insert should approximately be 1:3. The mixture was filled up with nuclease free water. Unless stated otherwise the reaction is incubated for 60 min at room temperature and is then used for transformation.

Gibson Assembly

Gibson assembly is a technique that allows fast isothermal assembly of multiple DNA fragments, regardless of fragment length. These fragments only need to have overlapping ends of 15-30 bp which can be created via PCR. Unless stated otherwise the Gibson Assembly was perfomed using the Gibson Assembly Cloning Kit (NEB) according to the protocol published by New England Biolabs. To design the primers for the respective PCRs a webtool was used (http://nebuilder.neb.com/).

  1. Set up the reaction according to the table below on ice (2-3 fragment assembly).
  2. Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
  3. Transform NEB 5-alpha Competent E. coli cells with 2 μl of the assembly reaction, following the transformation protocol.
Total Amount of Fragments 0.02-0.5 pmols
Gibson Assembly Master Mix (2X) 10 µl
Deionized H2O 10-X µl
Total Volume 20 µl

Transformation

The induction of competence of bacterial cells as well as the uptake of exogenous genetic material by these cells from their surroundings was done by using two different methods: Heatshock transformation and electroporation. The respective methods are listed below:

Heat Shock

  1. thaw cells on ice
  2. add 1 µl of plasmid DNA
  3. incubate on ice for 30 min
  4. heat shock at 42°C for 60 s
  5. incubate on ice for 5 min
  6. add 200 µl of SOC media
  7. incubate at 37°C for 2 h
  8. plate 20  and 200 µl on plates supplemented with the appropiate antibiotic

Electroporation

  1. add 1 μl plasmid to electrocompetent cells
  2. put DNA/ cell suspension in electroporation cuvette
  3. wipe dry the electroporator
  4. use a small plastic pipette to place the cells
  5. pulse: 2.5 kV, 200-400 Ω, 25 μF (for E.coli)
  6. immediatly add 1 ml LB and incubate for 2 h at 37°C
  7. plate 50 μl on selective medium plate
  8. centrifuge the rest (3000 g, 20 min), discard supernatant, re-suspend the pellet in 50 μl LB and plate it on selective medium plate

PCR

In addition to the common PCR for amplification of certain DNA fragments, several different types of PCR were used throughout our project. The purpose, procedure and generic use are listed in the table below.

Name Purpose Procedure Generic use Notes
colony/check PCR Check on insert length/ correct integration of insert primers binding in the vector upstream and downstream of the insert or one in insert and one in vector are used general reaction setup and procedure shown in tables below
gradient PCR Finding optimal conditions for our primers to bind and the PCRs in general Several PCRs batches are run within the same thermocycler, differing in annealing temperature August 6th
touchdown PCR avioding primer binding to non-specific sequences The annealing temperature is lowered successively with each cycle
SOE PCR side directed mutagenesis primers containing the desired base pair exchange/deletion/insertion are designed and used April 23th Splicing by Overlapping Extension PCR
QuikChange side directed mutagenesis general procedure by Agilent August 5th It was conducted by Vera at the laboratories of the Schwaneberg Group with supervision by Dr. rer. nat. Ljubica Vojcic.
general reaction procedure of a colony/check PCR
parameter duration temp [°C]
denature5:0095
anneal00:3056 30 cycles
elongate01:00 per kb72
denature00:3095
elongate05:0072
storeforever8
general reaction setup of a colony/check PCR
volume component
12.5 µlGoTaq Master Mix
1 µlprimer_F
1 µlprimer_R
9.5 µlddH2O
pick colony with tip and suspend in PCR tube