Team:Illinois/Antibody Receptor Tyrosine Kinase Fusion Notebook

From 2008.igem.org

(Difference between revisions)
(MfEQCIlenroHQYZG)
 
(68 intermediate revisions not shown)
Line 1: Line 1:
-
{{bottom_template}}
+
mZgAkl  <a href="http://fbdnisdgysij.com/">fbdnisdgysij</a>, [url=http://jzowycxvfekb.com/]jzowycxvfekb[/url], [link=http://lkctxlgmquum.com/]lkctxlgmquum[/link], http://obyjjyzxsbxr.com/
-
 
+
-
 
+
-
<!--PLACE TEXT BELOW HERE -->
+
-
== [https://2008.igem.org/Illinois/1_July_2008 July 1, 2008] ==
+
-
Made Yeast Media
+
-
 
+
-
YPD Medium, per liter:
+
-
 
+
-
10g yeast extract
+
-
 
+
-
20g peptone
+
-
 
+
-
20g dextrose
+
-
 
+
-
20g agar(only for plates)
+
-
 
+
-
1. Dextrose filter sterilized, the rest autoclaved
+
-
 
+
-
2. Weigh nutrients into flask double the volume you want to make, and stir to dissolve
+
-
 
+
-
3. Dextrose added to autoclaved media to equivalent of 20 g/L
+
-
 
+
-
4. Liquid media placed on bench, plate media placed in 65 degree water bath approximately 5 minutes
+
-
 
+
-
5. Poured into plates and allowed to solidify
+
-
 
+
-
 
+
-
== [https://2008.igem.org/Illinois/17_July_2008 July 17, 2008] ==
+
-
E.Coli Media
+
-
 
+
-
LB medium, per liter
+
-
 
+
-
10g tryptone
+
-
 
+
-
5g yeast extract
+
-
 
+
-
5g NaCl
+
-
 
+
-
1 mL 1N NaOH
+
-
 
+
-
15g (agar for plates)
+
-
 
+
-
1. Antibody DNA resuspended in TE buffer, 0.1 μg/μL
+
-
 
+
-
2. 5mL LB inoculated with single colony DH5a pro
+
-
 
+
-
3. Incubated at 37 degrees overnight
+
-
 
+
-
 
+
-
== [https://2008.igem.org/Illinois/18_July_2008 July 18, 2008] ==
+
-
Competent cells:
+
-
 
+
-
1. 3mL overnight culture of DH5a pro
+
-
 
+
-
2. Inoculated into 35mL of LB
+
-
 
+
-
3. OD600 checked, want 0.2-0.3
+
-
 
+
-
4. Place culture on ice for 3 minutes
+
-
 
+
-
5. Spin at 10,000 rpm for 7 minutes, discard supernatant
+
-
 
+
-
6. Resuspend in 10 mL cold 30 mM CaCl2, incubated on ice overnight
+
-
 
+
-
 
+
-
== [https://2008.igem.org/Illinois/19_July_2008 July 19, 2008] ==
+
-
Glycerol added to 10%, cells in freezer
+
-
 
+
-
Transformation:
+
-
 
+
-
1. Cells put in ice 30 min with 1μL plasmid(100ng)
+
-
 
+
-
2. Heat Shock for 2 minutes at 42 degrees
+
-
 
+
-
3. Ice for 8 minutes
+
-
 
+
-
4. Add cells to 1μL LB
+
-
 
+
-
5. Grow for 1 hour
+
-
 
+
-
6. Plate 100 to 200 μL on LB and amp, grow overnight
+
-
 
+
-
 
+
-
== [https://2008.igem.org/Illinois/21_July_2008 July 21, 2008] ==
+
-
Observations:
+
-
 
+
-
Plenty of colonies on all plates
+
-
 
+
-
Also, making 5mL overnight cultures for mini-prep tomorrow.
+
-
 
+
-
 
+
-
== July 22, 2008 ==
+
-
Mini spin prep on LC and HC colonies
+
-
 
+
-
Place DNA in 100μL H2O in freezer
+
-
 
+
-
 
+
-
== July 29, 2008 ==
+
-
All primers brought to standard concentration, 30mM
+
-
 
+
-
33.3μL H2O added per n mole of primer
+
-
 
+
-
 
+
-
== July 30, 2008 ==
+
-
Assembly PCR: amplify antibody gene fragments and then assemble them, light and heavy chains
+
-
 
+
-
{| class="wikitable" border="1"
+
-
|-
+
-
|tube
+
-
|1
+
-
|2
+
-
|3
+
-
|4
+
-
|5
+
-
|6
+
-
|7
+
-
|8
+
-
|-
+
-
|Antibody chain
+
-
|H
+
-
|H
+
-
|L
+
-
|L
+
-
|H
+
-
|H
+
-
|L
+
-
|L
+
-
|-
+
-
|DNA source
+
-
|colspan="4"|mini-prep
+
-
|colspan="4"|synthesized IDT genes
+
-
|-
+
-
|template
+
-
|colspan="8"|0.5ul
+
-
|-
+
-
|primers
+
-
|colspan="8"|1ul of appropriate forward and reverse primer
+
-
|-
+
-
|MgCl2
+
-
|3ul
+
-
|5ul
+
-
|3ul
+
-
|5ul
+
-
|3ul
+
-
|5ul
+
-
|3ul
+
-
|5ul
+
-
|-
+
-
|master mix
+
-
|colspan="8"|20ul
+
-
|-
+
-
|H20
+
-
|colspan="8"|to 50ul total volume
+
-
|}
+
-
 
+
-
Master mix already contains MgCl2; should have added extra to some tubes.
+
-
 
+
-
PCR program: 1. 94 degrees 5 min
+
-
2. 94 degrees 1 min
+
-
3. 50 degrees 1 min
+
-
4. 72 degrees 1 min
+
-
5. GOTO 2. 39 cycles
+
-
6. HOLD at 4 degrees
+
-
 
+
-
1.5% agarose gel made, 0.75g agarose in 50ml 0.5X TBE w/10ul EtBr.
+
-
 
+
-
20ul of PCR produts loaded on gel w/4ul 6X loading buffer, run at 200V for about half an hour.
+
-
 
+
-
All lanes had lots of DNA at ~375bp, PCR worked.
+
-
[[Image:07-30-08_AbFrag_Gel1.jpg|none]]
+
-
 
+
-
 
+
-
== July 31, 2008 ==
+
-
PCR to asemble fragments from yesterday.
+
-
 
+
-
{| class="wikitable" border="1"
+
-
|-
+
-
|tube
+
-
|1
+
-
|2
+
-
|3
+
-
|4
+
-
|-
+
-
|template from 7-30 PCR
+
-
|2+3
+
-
|2+7
+
-
|6+7
+
-
|1+8
+
-
|-
+
-
|template
+
-
|colspan="4"|0.5ul of both heavy and light chains
+
-
|-
+
-
|primers
+
-
|colspan="8"|1ul of appropriate forward and reverse primer
+
-
|-
+
-
|MgCl2
+
-
|0ul
+
-
|0ul
+
-
|3ul
+
-
|3ul
+
-
|-
+
-
|master mix
+
-
|colspan="4"|20ul
+
-
|-
+
-
|H20
+
-
|colspan="4"|to 50ul total volume
+
-
|}
+
-
 
+
-
PCR program: 1. 94 degrees 5 min
+
-
2. 94 degrees 1 min
+
-
3. 50 degrees 1 min
+
-
4. 72 degrees 1 min
+
-
5. GOTO 2. 29 cycles
+
-
6. HOLD at 4 degrees
+
-
 
+
-
Products run on a 1.5% agarose gel, no products of 700-800bp.
+
-
 
+
-
[[Image:7_31_08_assembly.jpg|none]]
+
-
 
+
-
 
+
-
== August 1, 2008 ==
+
-
PCR troubleshooting of yesterday's reaction:
+
-
use gradient for annealing temp (40-65 degrees), use more template, leave out end primers
+
-
 
+
-
Four series of reactions using a gradient annealing temp were run in addition to a fifth series omitting the end primers.
+
-
 
+
-
{| class="wikitable" border="1"
+
-
|-
+
-
|reaction series
+
-
|A
+
-
|B
+
-
|C
+
-
|D
+
-
|E
+
-
|-
+
-
|template (5+7) from 7-30 PCR
+
-
|0.5ul
+
-
|1ul
+
-
|1.5ul
+
-
|2ul
+
-
|1ul
+
-
|-
+
-
|primers
+
-
|colspan="5"|1ul of forward and reverse primers (omitted in E series)
+
-
|-
+
-
|master mix
+
-
|colspan="5"|20ul
+
-
|-
+
-
|H20
+
-
|colspan="5"|to 50ul total volume
+
-
|}
+
-
 
+
-
annealing temps: 40, 43.8, 50, 54, 60, and 64 degrees
+
-
 
+
-
PCR program: 1. 94 degrees 5 min
+
-
2. 94 degrees 1 min
+
-
3. annealing step 1 min
+
-
4. 72 degrees 1 min
+
-
5. GOTO 2. 29 cycles
+
-
6. HOLD at 4 degrees
+
-
 
+
-
PCR products run on 1.5% agarose gel at 200V for ~20 minutes, then 240V for ~20 minutes.
+
-
 
+
-
Products in the 700-800bp range in all lanes.  Perhaps it worked because of using a differnt product from the 7-30 PCR.
+
-
 
+
-
[[Image:8_1_08_assembly_gradient.jpg|none]]
+
-
 
+
-
 
+
-
== August 6, 2008 ==
+
-
1.5% agarose gel of 8-1 PCR products run to cut out bands. Products A1, B1, C1, and E1 used.
+
-
 
+
-
Bands around 800bp cut out for all lanes and invitrogen gel extraction kit used to collect DNA in 50ul H2O.
+
-
 
+
-
 
+
-
== September 5, 2008 ==
+
-
Flk-1 clones from [http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=MGC-18600&Template=mgcMouseClones ATCC] streaked onto LB+amp and incubated at 37 degrees.
+
-
 
+
-
 
+
-
== September 6, 2008 ==
+
-
Colonies picked from flk-1 plate and inoculated into 5ml LB+amp, grown at 37 degrees.
+
-
 
+
-
Freeze-dried e. coli with YCp50-poly from [http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=87555&Template=vectors ATCC] resuspended in 5ml LB, grown at 37 degrees.
+
-
 
+
-
 
+
-
== September 7, 2008 ==
+
-
Mini spin prep of flk-1 and YCp50-poly cells, plasmids stored in 50ul H2O.
+
-
 
+
-
 
+
-
 
+
-
 
+
-
<!-- == Insert Date Here ==
+
-
* lab procedure
+
-
** more lab procedure
+
-
* second lab procedure
+
-
** more about second lab procedure
+
-
COPY/PASTE THEN REMOVE THIS AND BRACKETS-->
+
-
 
+
-
{{bottom_template}}
+

Latest revision as of 06:22, 29 March 2016

mZgAkl <a href="http://fbdnisdgysij.com/">fbdnisdgysij</a>, [url=http://jzowycxvfekb.com/]jzowycxvfekb[/url], [link=http://lkctxlgmquum.com/]lkctxlgmquum[/link], http://obyjjyzxsbxr.com/

Retrieved from "http://2008.igem.org/Team:Illinois/Antibody_Receptor_Tyrosine_Kinase_Fusion_Notebook"