Team:METU/Protocols
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<table id="toc" class="toc"> | <table id="toc" class="toc"> | ||
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Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> | ||
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif""> | + | font-family:"Times New Roman","serif"">MgCl<sub>2</sub><span style="mso-tab-count:2"> </span>3 |
µL<o:p></o:p></span></p> | µL<o:p></o:p></span></p> | ||
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Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> | ||
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif""> | + | font-family:"Times New Roman","serif"">dH<sub>2</sub>O<span style="mso-tab-count:2"> </span>39,8 |
µL<o:p></o:p></span></p> | µL<o:p></o:p></span></p> | ||
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<p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | <p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | ||
"Times New Roman","serif"">To complete to 50 µl, add <span class="SpellE">ddH<span style="font-family:"Cambria Math","serif";mso-bidi-font-family:"Cambria Math"">2</span>O</span>.<br> | "Times New Roman","serif"">To complete to 50 µl, add <span class="SpellE">ddH<span style="font-family:"Cambria Math","serif";mso-bidi-font-family:"Cambria Math"">2</span>O</span>.<br> | ||
- | 1- Keep it in <span class="SpellE">waterbath</span> for 2.5 hours at | + | 1- Keep it in <span class="SpellE">waterbath</span> for 2.5 hours at 37°C.<br> |
2- Run the samples on the gel.<span style="mso-spacerun:yes"> </span><o:p></o:p></span></p> | 2- Run the samples on the gel.<span style="mso-spacerun:yes"> </span><o:p></o:p></span></p> | ||
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<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif"">Calculating | <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif"">Calculating | ||
Insert Amount:<o:p></o:p></span></b></p> | Insert Amount:<o:p></o:p></span></b></p> | ||
- | <img src="https://static.igem.org/mediawiki/2012/c/cb/K%C3%BCbra.png"> | + | <img src="https://static.igem.org/mediawiki/2012/c/cb/K%C3%BCbra.png"/> |
<p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | <p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | ||
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<li class="MsoNormal" style="mso-list:l6 level1 lfo19;background:white"><span lang="DE" style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif"; | <li class="MsoNormal" style="mso-list:l6 level1 lfo19;background:white"><span lang="DE" style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif"; | ||
mso-fareast-font-family:"Times New Roman";mso-ansi-language:DE;mso-fareast-language: | mso-fareast-font-family:"Times New Roman";mso-ansi-language:DE;mso-fareast-language: | ||
- | DE">Solve in | + | DE">Solve in ddH<sub>2</sub>0<o:p></o:p></span></li> |
</ul> | </ul> | ||
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Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> | ||
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Add 350 µL N3 buffer (cold <span class="GramE">+ | + | font-family:"Times New Roman","serif"">Add 350 µL N3 buffer (cold <span class="GramE">+4°C) inverse</span> gently and immediately.<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
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</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Spin down the cells at 10000g for 15 min | font-family:"Times New Roman","serif"">Spin down the cells at 10000g for 15 min | ||
- | at + | + | at +4°C.<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
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Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> | ||
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">0,1M | + | font-family:"Times New Roman","serif"">0,1M CaCI<sub>2</sub><span style="mso-spacerun:yes"> |
</span><o:p></o:p></span></p> | </span><o:p></o:p></span></p> | ||
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</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Divide 100 ml sample into 2 falcon and | font-family:"Times New Roman","serif"">Divide 100 ml sample into 2 falcon and | ||
- | centrifuge for 10 min at 5000 rpm at + | + | centrifuge for 10 min at 5000 rpm at +4°C.<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
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Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> | ||
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Add 10 ml 0.1 M cold | + | font-family:"Times New Roman","serif"">Add 10 ml 0.1 M cold CaCl<sub>2</sub> into |
supernatant and dissolve the pellet. Put it on ice for 10 min.<o:p></o:p></span></p> | supernatant and dissolve the pellet. Put it on ice for 10 min.<o:p></o:p></span></p> | ||
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</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Put 2 ml | + | font-family:"Times New Roman","serif"">Put 2 ml CaCl<sub>2</sub> and dissolve the pellet. <o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpLast" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpLast" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
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</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Put it on ice for 5 min and keep it at | font-family:"Times New Roman","serif"">Put it on ice for 5 min and keep it at | ||
- | + | + | +4°C.<o:p></o:p></span></p> |
<p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | <p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | ||
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background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif""><a href="http://www.neb.com/nebecomm/products/productm0202.asp" title="http://www.neb.com/nebecomm/products/productm0202.asp"><span style="color:windowtext;text-decoration:none;text-underline:none">Ligate | font-family:"Times New Roman","serif""><a href="http://www.neb.com/nebecomm/products/productm0202.asp" title="http://www.neb.com/nebecomm/products/productm0202.asp"><span style="color:windowtext;text-decoration:none;text-underline:none">Ligate | ||
- | + | 16°C/30 min, heat kill 80°C/20 min<o:p></o:p></span></a></span></li> | |
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
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"Times New Roman","serif""><a href="http://www.neb.com/nebecomm/products/productm0202.asp" title="http://www.neb.com/nebecomm/products/productm0202.asp"><span style="color:windowtext;text-decoration:none;text-underline:none"><o:p> </o:p></span></a></span></p> | "Times New Roman","serif""><a href="http://www.neb.com/nebecomm/products/productm0202.asp" title="http://www.neb.com/nebecomm/products/productm0202.asp"><span style="color:windowtext;text-decoration:none;text-underline:none"><o:p> </o:p></span></a></span></p> | ||
- | <p id="ref" class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""><a href="http://www.neb.com/nebecomm/products/productm0202.asp" title="http://www.neb.com/nebecomm/products/productm0202.asp"><span style="color:windowtext;text-decoration:none;text-underline:none"> | + | <p id="ref" class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""><a href="http://www.neb.com/nebecomm/products/productm0202.asp" title="http://www.neb.com/nebecomm/products/productm0202.asp"><span style="color:windowtext;text-decoration:none;text-underline:none">References:<o:p></o:p></span></a>12.</span></b></p> |
<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""><a href="http://www.neb.com/nebecomm/products/productm0202.asp" title="http://www.neb.com/nebecomm/products/productm0202.asp"><span style="color:windowtext;text-decoration:none;text-underline:none"><o:p> </o:p></span></a></span></b></p> | <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""><a href="http://www.neb.com/nebecomm/products/productm0202.asp" title="http://www.neb.com/nebecomm/products/productm0202.asp"><span style="color:windowtext;text-decoration:none;text-underline:none"><o:p> </o:p></span></a></span></b></p> | ||
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"Times New Roman","serif""><a href="http://www.neb.com/nebecomm/products/productm0202.asp" title="http://www.neb.com/nebecomm/products/productm0202.asp"><span style="color:windowtext;text-decoration:none;text-underline:none"><o:p> </o:p></span></a></span></p> | "Times New Roman","serif""><a href="http://www.neb.com/nebecomm/products/productm0202.asp" title="http://www.neb.com/nebecomm/products/productm0202.asp"><span style="color:windowtext;text-decoration:none;text-underline:none"><o:p> </o:p></span></a></span></p> | ||
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Revision as of 17:42, 25 September 2012
Protocols[gizle] |
1.PCR:
1.1.Colony PCR:
Prepare mastermix
according to your sample amount (except Taq and
template)
(For 1x sample preparation)
·
dNTP
1 µL
·
Buffer
5 µL
·
MgCl2 3
µL
·
**Forward Primer 0,5 µL
·
**Reverse Primer 0,5 µL
·
Template -
·
Taq
Polymerase 0,2 µL
·
dH2O 39,8
µL
·
TOTAL 50
µL
* You should add ingredients from largest amount to
smallest amount.
* Before addition of primers and template you can do
vortex.
** First you should pour ddH20 onto dried primers
according to the amount written in the primer sheet then you should dilute
(1:10) it in to new eppendorf. (10 ml primer+ 90 ml
ddH20)
Pour your samples into PCR tubes and add template
that you pick from the petri with toothpick or tip and finally add taq polymerase.
Then place your samples into the PCR machine and do
regular PCR.
1.2
PCR:
For a 25ul rxn:
·
Use 1ul of 60ng/ul
or 100ng/ul DNA
·
Use 1ul of each primer at 3.2pmole/ul concentration or 1.25ul of each primer at 100ng/ul concentration
·
2.5ul 10x PCR Buffer w/ Mg (1.5mM)
·
0.5ul 25mM MgCl2
·
0.5ul dNTP
·
0.125ul Taq
·
18.375ul sterile water to equal a 25ul rxn
(*if not making master mix, dilute Taq so that
you can add 1ul of Taq and 17.5ul
sterile water to equal a 25ul rxn)
For a 50ul rxn:
- Use
2ul of 60ng/ul or 100ng/ul
DNA
- Use
2ul of each primer at 3.2pmole/ul concentration
or 2.5ul of each primer at 100ng/ul
concentration
- 5ul
10x PCR Buffer w/ Mg
- 1ul
25mM MgCl2
- 1ul
dNTP
- 0.25ul
Taq
- 36.75ul
sterile water to equal a 50ul rxn
(*if not making a master mix, dilute Taq so
that you can add 1ul of Taq and 36ul
sterile water to equal a 50ul rxn)
- Keep
the reagents on ice.
- Add
the Taq last, and keep it in the freezer until
you are ready to add it.
- Vortex
briefly and quick spin.
- Cycle:
·
95°C for 1-5minutes
(usually 4min)
·
95°C for 1min
·
55°C for 1min
Cycle 30 times
·
72°C for 1.5 to
2min (usually 2min)
·
72°C for 10min
·
4°C hold
2.CULTURES:
2.1 Liquid Culture:
·
5 ml LB is mixed with appropriate
antibiotic and a culture tube is prepared with certain labels.
·
A pipette tip is used to pick up a
colony from the plate and release it into the LB by stirring and pipetting up
and down
·
Incubate the culture at 37°C for 13-15
hours. Do not let the cells become old to not release their plasmids
·
If the culture becomes saturated: you
can reinoculate 30µL into a new 3mL LB tube
2.2 Glycerol Stock:
·
Put 500µL of a mid-log culture into a
1.5mL tube with 500µL 80% glycerol
·
Store at -80C
2.3 Spreading Plates:
·
Put 50 µL and 150 µL of whether LB
culture or transformed cells.
·
Spread the cells into the plate until
there is no seen liquid.
·
Wait until the plates dried.
·
After 14-16 hours colonies will show up.
2.4 Streaking Plates:
·
Put a drop your culture on the plate
·
Using a pipette tip,
spread the drop out into a zigzag. Then use one edge of the zigzag to draw out
another zigzag. Repeat to have about 3 zigzags (this makes the culture get
spread out more and more with each streak)
·
Wait until the plates dried.
·
After 14-16 hours colonies will show up.
3.GELS:
3.1
Gel Preparation (%1 gel)
·
Add 0,5 gr agarose to 50 ml TAE buffer.
·
Until agorose
being melted, heat it in microwave and do not forget to stir it often.
·
After melting, it should be cool enough
to handle it.
·
Add 5 ml EtBr.
·
Then, pour it to container and pay
attention not to form bubbles in gel.
3.2
Gel Photo Imaging:
·
Adjust zoom and position using visible
light
·
Before turning on UV load your settings
file which has the following parameters:
·
Preview tab, all three options checked
·
Active image
·
Dynamic integration, auto exposure, 10
frames
·
50/50 brightness/contrast
·
Maximize brightness with camera knob
(counterclockwise)
·
Turn on UV light
·
Lower brightness from camera knob if
necessary
4.GETTING THE DNA PARTS FROM KIT PLATE:
·
10 ul ddHO is added into the kit plate target part.
·
Wait 5 min.and
get the part by well pipetting.
5.TRANSFORMATION:
Materials:
·
Resuspended
DNA (2 ul )
·
Competent
cells (50ul per transformation)
·
Ice
·
2ml tube (1 per a transformation')
·
42ºC water bath
·
Petri dishes with LB agar and
appropriate antibiotic (2 or 3 per transformation)
·
Glass beads or spreader
·
37ºC incubator
Preparation:
·
Start thawing the competent cells on
ice.
·
Add 50 µL of thawed competent cells into
pre-chilled 2ml tube.
·
Add
1 - 2 µL of the resuspended DNA to the 2ml tube. Pipet
up and down a few times, gently. Make sure to keep the competent cells on ice.
·
Close tube and incubate the cells on ice
for 30 minutes.
·
Heat shock
the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
·
Incubate the cells on ice for 5 minutes.
·
Add 400 µl
of LB media (make sure that the broth does not contain antibiotics and is not
contaminated)
·
Incubate the cells at 37ºC for 2 hours
while the tubes are rotating or shaking. Important: 2 hour recovery time helps
in transformation efficiency, especially for plasmid backbones with antibiotic
resistance other than ampicillin.
·
Label two petri dishes with LB agar and
the appropriate antibiotic(s) with the part number, plasmid backbone, and
antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the
dishes, and spread. This helps ensure that you will be able to pick out a
single colony.
·
Incubate the plate at 37ºC for 12-14
hours, making sure the agar side of the plate is up. If incubated for too long
the antibiotics start to break down and un-transformed cells will begin to
grow. This is especially true for ampicillin - because the resistance enzyme is
excreted by the bacteria, and inactivates the antibiotic outside of the
bacteria.
·
You can pick a single colony, make a
glycerol stock, grow up a cell culture.
6.RESTRICTION DIGESTION:
NEB Buffer 5 µl
BSA 0.5 µl
Enzyme 1 0.5 µl
Enzyme 2 0.5 µl
Plasmid* à
1000ng/ml
To complete to 50 µl, add ddH2O.
1- Keep it in waterbath for 2.5 hours at 37°C.
2- Run the samples on the gel.
7.LIGATION:
·
10X T4 ligase buffer: 2.0 µL
·
6:1 molar ratio of insert to vector
(~10ng vector)
·
Add (8.5 - vector and insert volume)µl ddH2O
·
T4 Ligase: 1 µL
·
Incubation at room temperature 1 hour
and 15 min at 65°C for enzyme inactivation.
·
Alternatively: Incubation at +4°C
overnight.
Calculating
Insert Amount:
![](https://static.igem.org/mediawiki/2012/c/cb/K%C3%BCbra.png)
8.MATERIALS AND CHEMICALS:
8.1 TAE Buffer:
For 1 L of 50 x TAE buffer you need:
- 242.48 g Tris
- 41.02 g Sodiumacetate
- 18.612 g EDTA
- Adjust pH to 7.8
- Solve in dH2O
20 mL of the stock is diluted in 1 L
dH2O for the gel electrophoresis.
8.2 DNA Loading Buffer
- 50 % (v/v) glycerol
- 1 mM EDTA
- 0.1 % (w/v) bromphenol blue
- Solve in ddH20
8.3 LB Medium
For 1 L of LB medium you need:
- 10 g Trypton
- 5 g yeast extract
- 10 g NaCl
- 12 g
Agar-Agar (for plates)
- Adjust pH to 7.0
8.4
LB Agar
For 1 L of LB Agar you need
- 10 g Trypton
- 5 g yeast extract
- 10 g NaCl
- 12 g
Agar-Agar (for plates)
- Adjust pH to 7.0
9.QIAGEN Kits
9.1
Plasmid Isolation:
Usually there is a protocol in the kit. First, you should
read it.
·
Take 2 ml cell in to the eppendorf (2ml).
·
Spin down the cells at 10000g for 1 min.
·
Discard supernatant.
·
Add 2 ml cell
in to the eppendorf again, totally 4 ml will be
pelleted.
·
Spin down the cells at 10000g for 1 min.
·
Discard supernatant.
·
Resuspend
the cells with 250 µL p1* buffer and vortex.
·
Add 250 µL P2 buffer and make sure the
blue color is equally spread. Gently inverse the eppendorf
4-5 times. DON’T VORTEX!!!
·
Wait 5 min at room temperature.
·
Add 350 µL N3 buffer (cold +4°C) inverse gently and immediately.
·
Incubate on ice for 15 min.
·
Spin down the cells at 10000g for 15 min
at +4°C.
·
Place the supernatant into the tube that
is placed into the kit.
·
Spin down the cells at 10000g for 1 min.
·
Remove the below part of the tube and
discard supernatant.
·
Add 500 µL PB buffer.
·
Add 750 µL PE buffer and wait 1 min near
the flame.
·
Spin down the cells at 10000g for 1 min.
·
Spin down the cells at 13000g for 1 min
again.
·
Wait 5 min near the flame.
·
Put the filter parts onto the 1,5 ml eppendorfs.
·
Add 50 µL elution buffer.
·
Wait 1 min near the flame.
·
Spin down the cells at 13000g for 1 min.
·
Store your plasmids at+4°C for 1 hour
before further experiments.
9.2
Gel Extraction:
We used QIAGEN kit do with the following
changes:
http://kirschner.med.harvard.edu/files/protocols/QIAGEN_QIAquickSpin_EN.pdf
Changes
in that protocol:
At step 0: Wear
disposable lab coat or long sleeves to prevent UV-burn. Use a razor
blade/scalpel to excise the band.
At step 4: Add 10 ul for
3M NaOAc no matter if the color is yellow or not.
This will correct the pH and should turn the QG back to yellow.
At step 7: Combine samples of
identical DNA in the same column (spin multiple times if necessary).
At step 13: Elute in 30 ul
rather than 50 ul to really maximize yield.
9.3
PCR Purification
We used the protocol provided with the QIAGEN kit.
·
3 volumes of Buffer QG is added to 1
volume of the PCR sample and mixed by vortexing.
·
1 volume of the INITIAL sample volume of
isopropanol is added to the sample and mix.
·
Place a QIAquick
spin column in a provided 2 ml collection tube. Apply the sample to the QIAquick column with the pipette and centrifuge for 30–60 s.Discard flow-through. Place the QIAquick
column back into the same tube
·
Add 0.75 ml Buffer PE to the QIAquick column with the pipette and centrifuge for 30–60
s. Discard flow-through and place the QIAquick column
back in the same tube.
·
Centrifuge the column for an additional
1 min.
·
Label the top of clean 1.7 ml microcentrifuge tube(s) with the name of your sample(s).
Transfer QIAquick column(s) to the tube(s).
·
To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) to the center
of the QIAquick membrane. Let it stand for 1 min and centrifuge
the column for 1 min.
10.Competent Cells:
Materials:
·
Detergent-free, sterile glassware and plasticware
·
Table-top OD600nm spectrophotometer
·
1 ml DH5 alpha cells
·
LB
·
0,1M CaCI2
Preparation:
·
Take 1ml from the DH5 alpha cells which
were grown one day ago and put it in to 100ml LB medium. Keep it at 370C for 2
hours and measure it by spectrophotometer at 600 nm wavelength until absorbance
reaches OD of
0.375.
·
Divide 100 ml sample into 2 falcon and
centrifuge for 10 min at 5000 rpm at +4°C.
·
Discard the supernatant.
·
Add 10 ml 0.1 M cold CaCl2 into
supernatant and dissolve the pellet. Put it on ice for 10 min.
·
Centrifuge at 5000 rpm for 5 min at
+40C. Then, discard the supernatant.
·
Add 10 ml CaCl2 and dissolve it by
shaking it up and down.
·
Put the sample on ice for half an hour.
·
Centrifuge at 5000 rpm for 5 min at +4°C
.Then, discard the supernatant.
·
Put 2 ml CaCl2 and dissolve the pellet.
·
Put it on ice for 5 min and keep it at
+4°C.
11.
3A Assembly Kit:
We used the linear plasmids and the procedure that
the iGEM provides us;
11.1
Digestion:
- Enzyme Master Mix for Plasmid
Backbone (25ul total, for 6 rxns)
- 5 ul NEB
Buffer 2
- 0.5 ul
BSA
- 0.5 ul EcoRI-HF
- 0.5 ul PstI
- 0.5 ul DpnI (Used to digest any template DNA
from production)
- 18 ul dH20
- Digest
Plasmid Backbone
- Add 2ul
of digested plasmid backbone (25 ng)
- Add equimolar amount of EcoRI-HF
SpeI digested fragment (< 3 ul)
- Add equimolar amount of XbaI PstI digested fragment (< 3 ul)
- Add 1 ul T4
DNA ligase buffer.
- Add 0.5 ul T4
DNA ligase
- Add
water to 10 ul
- Ligate
16°C/30 min, heat kill 80°C/20 min
- Transform
with 1-2 ul of product
References: