Team:HIT-Harbin/Protocol

From 2013.igem.org

(Difference between revisions)
 
(One intermediate revision not shown)
Line 27: Line 27:
                 <div class="mainarea">
                 <div class="mainarea">
                     <div class="multi-columns">
                     <div class="multi-columns">
-
                         <div class="half">
+
                         <div class="third">
                             <h4></h4>1.The composition of LB medium for E. coli
                             <h4></h4>1.The composition of LB medium for E. coli
                             <p></p>1)Liquid medium (1L)
                             <p></p>1)Liquid medium (1L)
Line 42: Line 42:
                             <p></p>Sterilize the mixture in autoclave at 121℃ for 30 minutes.
                             <p></p>Sterilize the mixture in autoclave at 121℃ for 30 minutes.
                         </div>
                         </div>
-
                         <div class="half last">
+
                         <div class="third">
                             <h4></h4>2.The preparation of competent E.coli cell (DH5α)
                             <h4></h4>2.The preparation of competent E.coli cell (DH5α)
                             <p></p>1)pick one colony and put it in 3~5ml LB media , overnight at 37℃
                             <p></p>1)pick one colony and put it in 3~5ml LB media , overnight at 37℃
Line 53: Line 53:
                             <p></p>8)Remove the supernatant and resuspend cells (by pipetting) in 4 ml cold 0.1 M CaCl2 containing 15% glycerol. Transfer 100uL the mixture into 1.5 ml centrifuge tubes placed on ice. Cells are stored at -80℃ and can be used for transformation in 6 months
                             <p></p>8)Remove the supernatant and resuspend cells (by pipetting) in 4 ml cold 0.1 M CaCl2 containing 15% glycerol. Transfer 100uL the mixture into 1.5 ml centrifuge tubes placed on ice. Cells are stored at -80℃ and can be used for transformation in 6 months
                         </div>
                         </div>
-
                    </div>
+
                         <div class="third last">
-
 
+
-
                    <div class="multi-columns">
+
-
                         <div class="third">
+
                             <h4></h4>3.Transformation
                             <h4></h4>3.Transformation
<p></p>1)Thaw the competent cells on ice
<p></p>1)Thaw the competent cells on ice
Line 67: Line 64:
<p></p>8)Incubate the cells at 37℃, 110rpm for 1 hour
<p></p>8)Incubate the cells at 37℃, 110rpm for 1 hour
                         </div>
                         </div>
 +
                    </div>
 +
                    <div class="multi-columns">
                         <div class="third">
                         <div class="third">
                             <h4></h4>4.Restriction enzyme reaction system
                             <h4></h4>4.Restriction enzyme reaction system
Line 82: Line 81:
<p></p>The enzymes using in the experiments are all from Thermo company.
<p></p>The enzymes using in the experiments are all from Thermo company.
                         </div>
                         </div>
-
                         <div class="third last">
+
                         <div class="third">
                             <h4></h4>5. Ligation system reaction
                             <h4></h4>5. Ligation system reaction
<p></p>1)Prepare the following reaction system:
<p></p>1)Prepare the following reaction system:
Line 88: Line 87:
<p></p>2)Incubate it more than 1 hour at 22 ℃ water bath  
<p></p>2)Incubate it more than 1 hour at 22 ℃ water bath  
                         </div>
                         </div>
-
                    </div>
+
                         <div class="third last">
-
 
+
-
                    <div class="multi-columns">
+
-
                         <div class="fourth">
+
                             <h4></h4>6. PCR reaction
                             <h4></h4>6. PCR reaction
<p></p>1)Prepare the following reaction system:
<p></p>1)Prepare the following reaction system:
Line 98: Line 94:
<img src="https://static.igem.org/mediawiki/2013/4/43/Protocol4.png"/>
<img src="https://static.igem.org/mediawiki/2013/4/43/Protocol4.png"/>
                         </div>
                         </div>
-
                         <div class="fourth">
+
                    </div>
 +
                    <div class="multi-columns">
 +
                         <div class="third">
                             <h4></h4>7. Agarose Electrophoresis
                             <h4></h4>7. Agarose Electrophoresis
<p></p>1)Prepare a piece of 1% weight-to-volume agarose and add SYBR dye or ethidium bromide
<p></p>1)Prepare a piece of 1% weight-to-volume agarose and add SYBR dye or ethidium bromide
Line 107: Line 105:
<p></p>6)Run at 120V
<p></p>6)Run at 120V
                         </div>
                         </div>
-
                         <div class="fourth">
+
                         <div class="third">
                           <h4></h4>8. Purification of DNA
                           <h4></h4>8. Purification of DNA
<p></p>1)Cut out the DNA fragment from the agarose gel with a razor blade, while minimizing the size of the gel slice
<p></p>1)Cut out the DNA fragment from the agarose gel with a razor blade, while minimizing the size of the gel slice
Line 120: Line 118:
<p></p>10)Add 40 uL elution buffer to the center of the column and wait at least 2 minutes
<p></p>10)Add 40 uL elution buffer to the center of the column and wait at least 2 minutes
<p></p>11)Centrifuge it at 12,000 rpm for 1 minutes
<p></p>11)Centrifuge it at 12,000 rpm for 1 minutes
-
 
-
 
                         </div>
                         </div>
-
                        <div class="fourth last">
+
                    </div>
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum.</p>
+
                          
-
                        </div>
+
-
                    </div>
+
-
 
+
-
                    <div class="multi-columns">
+
-
                         <div class="fourth">
+
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.</p>
+
-
                        </div>
+
-
                        <div class="half">
+
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum. Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua.</p>
+
-
                        </div>
+
-
                        <div class="fourth last">
+
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.</p>
+
-
                        </div>
+
-
                    </div>
+
-
 
+
-
                    <div class="multi-columns">
+
-
                        <div class="half">
+
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum. Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua.</p>
+
-
                        </div>
+
-
                        <div class="fourth">
+
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.</p>
+
-
                        </div>
+
-
                        <div class="fourth last">
+
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.</p>
+
-
                        </div>
+
-
                    </div>
+
-
 
+
-
                    <div class="multi-columns">
+
-
                        <div class="fourth">
+
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.</p>
+
-
                        </div>
+
-
                        <div class="fourth">
+
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.</p>
+
-
                        </div>
+
-
                        <div class="half last">
+
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum. Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua.</p>
+
-
                        </div>
+
-
                    </div>
+
-
 
+
-
                    <div class="multi-columns">
+
-
                        <div class="third">
+
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua.</p>
+
-
                        </div>
+
-
                        <div class="twothirds last">
+
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.</p>
+
-
                        </div>
+
-
                    </div>
+
-
 
+
-
<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum</p>
+
-
<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum</p>
+
-
                  <h4 class="hashed"><span>Image positioning</span></h4>
+
-
 
+
-
                    <div class="preload alignleft">
+
-
                        <img src="https://static.igem.org/mediawiki/2013/2/27/099.png" alt="" />
+
-
                    </div>
+
-
                    <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum</p>
+
-
                    <p>Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem.</p>
+
-
 
+
-
                    <div class="separator"></div>
+
-
 
+
-
                    <div class="preload alignright">
+
-
                        <img src="https://static.igem.org/mediawiki/2013/2/27/099.png" alt="" />
+
-
                    </div>
+
-
                    <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum</p>
+
-
                    <p>Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem.</p>
+
-
 
+
</div>
</div>
</div>
</div>

Latest revision as of 01:35, 27 September 2013

HIT-Harbin

HIT-Harbin

Nootbook/Protocol

1.The composition of LB medium for E. coli

1)Liquid medium (1L)

NaCl 10g

Tryptone 10g

Yeast extract 5g

Distilled water 1000ml

2)Solid medium

NaCl 10g

Tryptone 10g

Yeast extract 5g

Distilled water 1000ml

Agar powder 10~20g

Sterilize the mixture in autoclave at 121℃ for 30 minutes.

2.The preparation of competent E.coli cell (DH5α)

1)pick one colony and put it in 3~5ml LB media , overnight at 37℃

2)Transfer the centrifugal sediment into 100ml LB liquid media

3)Grow the cuLture at 37℃(250rpm), until OD600 = 0.4 (2-3h)

4)Place cuLture on ice for 10 minutes

5)Centrifuge cuLture at 4℃ for 10 minutes at 4,000 rpm Subsequent resuspensions is done in the same tube. CuLture remains cold for the rest of procedure. Place tubes on ice and resuspend the cuLture the on ice

6)Remove the media and resuspend cells in 10ml cold 0.1 M CaCl2 and incubate it on ice for 30 minutes

7)Centrifuge it at 4℃ for 10 minutes at 3,500 rpm

8)Remove the supernatant and resuspend cells (by pipetting) in 4 ml cold 0.1 M CaCl2 containing 15% glycerol. Transfer 100uL the mixture into 1.5 ml centrifuge tubes placed on ice. Cells are stored at -80℃ and can be used for transformation in 6 months

3.Transformation

1)Thaw the competent cells on ice

2)Add 50 uL of thawed competent cells into pre-chilled 1.5ml tube

3)Add the total ligation plasmid into the 1.5 ml tube. Overturn gently the tube a few times. Make sure that the competent cells is always on ice

4)Close the tubes and incubate the mixture on ice for 30 minutes

5)Heat shock the cells by water bath at 42℃ for 45 seconds

6)Place the cells on ice for 1 minutes

7)Add 400 uL LB liquid media into the tubes

8)Incubate the cells at 37℃, 110rpm for 1 hour

4.Restriction enzyme reaction system

1)formula

2)Mix gently and centrifuge it for a few seconds

3)Incubate it at 37℃ for 5 minutes

The combination of double enzyme digestion are as following:

FD EcoRⅠ、FD Pst Ⅰ

FD XbaⅠ、FD Pst Ⅰ

FD EcoRⅠ、FD Spe Ⅰ

FD SpeⅠ、FD Pst Ⅰ

FD EcoR Ⅰ、FD Xba Ⅰ

The enzymes using in the experiments are all from Thermo company.

5. Ligation system reaction

1)Prepare the following reaction system:

2)Incubate it more than 1 hour at 22 ℃ water bath

6. PCR reaction

1)Prepare the following reaction system:

2)The PCR reaction setting:

7. Agarose Electrophoresis

1)Prepare a piece of 1% weight-to-volume agarose and add SYBR dye or ethidium bromide

2)Place the agarose in the apparatus rig with the wells facing the negative electrode (black-colored)

3)Fill the rig with 1x TAE buffer

4)Load 5 uL 1 kb marker

5)Add 1 uL of 6x buffer to 5 uL DNA sample. Load the mixture

6)Run at 120V

8. Purification of DNA

1)Cut out the DNA fragment from the agarose gel with a razor blade, while minimizing the size of the gel slice

2)Weigh the gel slice and add 3 volumes of gel lysis buffer to every 1 volume of gel(100mg = 100 uL)

3)Wait 2 minutes

4)Centrifuge it at 12,000 rpm for 1 minute

5)Discard the flow-through and repeat Step 4 until all sample has passed through the column

6)Add 700 uL rinse buffer into the column, then centrifuge it at 12,000 rpm for 1 minute

7)Discard the flow-through and add 500 uL rinse buffer into the column, then centrifuge it at 12,000 rpm for 1 minute

8)Discard the flow-through and centrifuge the column at 12,000 for 2 minutes

9)Transfer the column to a new centrifuge tube. Open the tube at 37℃ for a few minutes to volatilize the alcohol

10)Add 40 uL elution buffer to the center of the column and wait at least 2 minutes

11)Centrifuge it at 12,000 rpm for 1 minutes