User:Kleinsorg/test

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= 13/09/2010 =
 +
<br />
 +
'''Restriction digest''' of psiCHECK-2 plasmid <br />
 +
This will be used as backbone for raPCR cloning.  Enzymes: XhoI and NotI
 +
 
 +
Assay:
 +
:5 µL 10x NEBuffer 3
 +
:5 µL 10x BSA
 +
:5 µL plasmid (psiCHECK-2, ~370 ng/µL)
 +
:3 µL XhoI
 +
:1 µL NotI
 +
:18.6 µL H<sub>2</sub>O
 +
 
 +
Restriction digest was performed for approx. 5h
 +
 
 +
<br /><br />
 +
'''raPCR''' to create binding sites for different miRNAs
 +
This random assembly PCR (raPCR) will be done to create binding site patterns for the miRNAs mentioned. In the first PCR step the oligos will basically anneal and constructs of different lengths will form. In the second step, the stop oligos are used as primers to amplify the previously formed constructs.
 +
 
 +
* first tries are: hsa-mir-122, hsa-mir122(ran9-12) and mm-mir-376a/375 (Oligos: [[Igem2010/Main/Primer_database#Primer_Table_raPCR|ra001-003 and ra006]])<br />
 +
 
 +
stop-oligos: raPCR_AS13-stop_rev_NotI and  raPCR_AS13-stop_fw_XhoI ([[Igem2010/Main/Primer_database#Primer_Table_raPCR|ra017/018]])<br />
 +
spacer: raPCR_AS13-spacer(0) and raPCR_AS13-spacer(10)  ([[Igem2010/Main/Primer_database#Primer_Table_raPCR|ra012/013]])
 +
 
 +
Oligos were used in standard conc. (100µM)
 +
 
 +
*'''1. PCR'''
 +
{| border="1" cellpadding="3" style="border:solid 1px #AAAAAA; border-collapse:collapse; background-color:#F9F9F9; empty-cells:show;"
 +
! Oligo !! mir-122 !! mir-122(ran9-12) !! mir-375/376a
 +
|-
 +
|miR || 1 or 3 µL || 1 or 3 µL  || 0.5 or 1.5 µL (each)
 +
|-
 +
|spacer(0)or (10) || 1 µL || 1 µL || 1 µL
 +
|-
 +
|stop || 0 or 0.5 µL each || 0 or 0.5 µL each  || 0 or 0.5 µL each
 +
|-
 +
|}
 +
<br />
 +
Total: 12 reactions<br />
 +
each reaction was set up in 30 µL, using 2x Phusion Mastermix for 12 cycles
 +
 
 +
{| border="1" cellpadding="3" style="border:solid 1px #AAAAAA; border-collapse:collapse; background-color:#F9F9F9; empty-cells:show;"
 +
!Temp !!  Time !!
 +
|-
 +
|95°||05:00||
 +
|-
 +
|style="border-top:solid 1px #000000;"|95°||style="border-top:solid 1px #000000;border-right:solid 1px #000000;"|00:30||rowspan="3"|x 12 cycles
 +
|-
 +
|57°||style="border-right:solid 1px #000000;"|00:45
 +
|-
 +
|style="border-bottom:solid 1px #000000;"|72°||style="border-bottom:solid 1px #000000;border-right:solid 1px #000000;"|00:45
 +
|-
 +
|4° || forever ||
 +
|}
 +
 
 +
*'''PCR purification:''' each PCR was purified using Qiagen PCR purification Kit and eluted in 32 µL<br />
 +
for the next PCR, three assay will tried:<br />
 +
# 5µL eluate + 1 µL of each stop oligo in 50µL total volume
 +
# 5µL eluate + 2 µL of each stop oligo in 50µL total volume
 +
# 20µL eluate + 1 µL of each stop oligo in 50µL total volume<br /><br />
 +
stop-oligos: raPCR_AS13-stop_rev_NotI and  raPCR_AS13-stop_fw_XhoI<br />
 +
 
 +
*'''2. PCR'''
 +
In total there were 72 reactions. Each was run with 2x Phusion Mastermix, missing volume was filled with water.
 +
 
 +
{| border="1" cellpadding="3" style="border:solid 1px #AAAAAA; border-collapse:collapse; background-color:#F9F9F9; empty-cells:show;"
 +
!Temp !!  Time !!
 +
|-
 +
|95°||05:00||
 +
|-
 +
|style="border-top:solid 1px #000000;"|95°||style="border-top:solid 1px #000000;border-right:solid 1px #000000;"|00:30||rowspan="3"|x 25 cycles
 +
|-
 +
|65°||style="border-right:solid 1px #000000;"|00:45
 +
|-
 +
|style="border-bottom:solid 1px #000000;"|72°||style="border-bottom:solid 1px #000000;border-right:solid 1px #000000;"|00:50
 +
|-
 +
|72°||05:00||
 +
|-
 +
|4° || forever ||
 +
|}
 +
 
 +
DNA was stored in fridge afterwards
 +
 
 +
<br /><br /><br /><br />
 +
 
 +
----
 +
 
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{| cellpadding="5" cellspacing="0" style="text-align: center; color:#f3d675; border: 3px solid #4e93a4; margin-left:25px;"
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|- border="0"
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! colspan="7" style="background:#4e93a4;" | [[Igem2010/Main/Library/Notebook/July|<font color="#ffecba">July</font>]]
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|- style="background:#4e93a4; color:white"
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|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
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|-
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|colspan="3"| ||'''1'''||'''2'''||'''3'''||'''4'''
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|-
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|'''[[Igem2010/Main/Library/Notebook/July#05/07/2010 - 11/07/2010|5]]'''||'''[[Igem2010/Main/Library/Notebook/July#05/07/2010 - 11/07/2010|6]]'''||'''[[Igem2010/Main/Library/Notebook/July#05/07/2010 - 11/07/2010|7]]'''||'''[[Igem2010/Main/Library/Notebook/July#05/07/2010 - 11/07/2010|8]]'''||'''[[Igem2010/Main/Library/Notebook/July#05/07/2010 - 11/07/2010|9]]'''||'''[[Igem2010/Main/Library/Notebook/July#05/07/2010 - 11/07/2010|10]]'''||'''[[Igem2010/Main/Library/Notebook/July#05/07/2010 - 11/07/2010|11]]'''
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|-
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|'''[[Igem2010/Main/Library/Notebook/July#12/07/2010 - 19/07/2010|12]]'''||'''[[Igem2010/Main/Library/Notebook/July#12/07/2010 - 19/07/2010|13]]'''||'''[[Igem2010/Main/Library/Notebook/July#12/07/2010 - 19/07/2010|14]]'''||'''[[Igem2010/Main/Library/Notebook/July#12/07/2010 - 19/07/2010|15]]'''||'''[[Igem2010/Main/Library/Notebook/July#12/07/2010 - 19/07/2010|16]]'''||'''[[Igem2010/Main/Library/Notebook/July#12/07/2010 - 19/07/2010|17]]'''||'''[[Igem2010/Main/Library/Notebook/July#12/07/2010 - 19/07/2010|18]]'''
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|-
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|'''[[Igem2010/Main/Library/Notebook/July#12/07/2010 - 19/07/2010|19]]'''||'''[[Igem2010/Main/Library/Notebook/July#20/07/2010|20]]'''||'''[[Igem2010/Main/Library/Notebook/July#21/07/2010|21]]'''||'''[[Igem2010/Main/Library/Notebook/July#22/07/2010|22]]'''||'''[[Igem2010/Main/Library/Notebook/July#23/07/2010|23]]'''||'''24'''||'''25'''
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|-
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|'''[[Igem2010/Main/Library/Notebook/July#26/07/2010|26]]'''||'''[[Igem2010/Main/Library/Notebook/July#27/07/2010|27]]'''||'''[[Igem2010/Main/Library/Notebook/July#28/07/2010|28]]'''||'''29'''||'''30'''||'''[[Igem2010/Main/Library/Notebook/July#31/07/2010|31]]'''||
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|-
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| colspan="7"| <span style="color:#ffffff">-</span>
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|}
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<br>
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{| cellpadding="5" cellspacing="0" style="text-align: center; color:#f3d675; border: 3px solid #4e93a4; margin-left:25px;"
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|- border="0"
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! colspan="7" style="background:#4e93a4;" | [[Igem2010/Main/Library/Notebook/August|<font color="#ffecba">August</font>]]
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|- style="background:#4e93a4; color:white"
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|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
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|-
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|colspan="6"| ||'''[[Igem2010/Main/Library/Notebook/August#01/08/2010|1]]'''
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|-
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|'''[[Igem2010/Main/Library/Notebook/August#02/08/2010|2]]'''||'''[[Igem2010/Main/Library/Notebook/August#03/08/2010|3]]'''||'''[[Igem2010/Main/Library/Notebook/August#04/08/2010|4]]'''||'''[[Igem2010/Main/Library/Notebook/August#05/08/2010|5]]'''||'''[[Igem2010/Main/Library/Notebook/August#06/08/2010|6]]'''||'''[[Igem2010/Main/Library/Notebook/August#07/08/2010|7]]'''||'''[[Igem2010/Main/Library/Notebook/August#08/08/2010|8]]'''
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|-
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|'''[[Igem2010/Main/Library/Notebook/August#09/08/2010|9]]'''||'''[[Igem2010/Main/Library/Notebook/August#10/08/2010|10]]'''||'''[[Igem2010/Main/Library/Notebook/August#11/08/2010|11]]'''||'''12'''||'''13'''||'''14'''||'''15'''
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|-
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|'''16'''||'''17'''||'''18'''||'''19'''||'''20'''||'''21'''||'''22'''
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|'''23'''||'''24'''||'''25'''||'''26'''||'''27'''||'''28'''||'''29'''
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|-
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|'''30'''||'''31'''||colspan="5"|
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|}
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{| cellpadding="5" cellspacing="0" style="text-align: center; color:#f3d675; border: 3px solid #4e93a4;margin-left:25px;"
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|- border="0"
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! colspan="7" style="background:#4e93a4;" | [[Igem2010/Main/Library/Notebook/September|<font color="#ffecba">September</font>]]
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|- style="background:#4e93a4; color:white"
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|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
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|-
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|colspan="2"| ||'''1'''||'''2'''||'''3'''||'''4'''||'''5'''
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|-
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|'''6'''||'''7'''||'''8'''||'''9'''||'''10'''||'''11'''||'''12'''
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|-
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|'''[[Igem2010/Main/Library/Notebook/September#13/09/2010|13]]'''||'''[[Igem2010/Main/Library/Notebook/September#14/09/2010|14]]'''||'''[[Igem2010/Main/Library/Notebook/September#15/09/2010|15]]'''||'''[[Igem2010/Main/Library/Notebook/September#16/09/2010|16]]'''||'''[[Igem2010/Main/Library/Notebook/September#17/09/2010|17]]'''||'''[[Igem2010/Main/Library/Notebook/September#18/09/2010|18]]'''||'''19'''
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|-
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|'''20'''||'''21'''||'''22'''||'''23'''||'''24'''||'''25'''||'''26'''
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|-
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|'''27'''||'''28'''||'''29'''||'''30'''||colspan="3"|
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|-
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| colspan="7"| <span style="color:#ffffff">-</span>
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<br>
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{| cellpadding="5" cellspacing="0" style="text-align: center; color:#f3d675; border: 3px solid #4e93a4;margin-left:25px;"
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|- border="0"
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! colspan="7" style="background:#4e93a4;" | [[Igem2010/Main/Library/Notebook/October|<font color="#ffecba">October</font>]]
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|- style="background:#4e93a4; color:white"
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|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
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|'''4'''||'''5'''||'''6'''||'''7'''||'''8'''||'''9'''||'''10'''
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|-
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|'''11'''||'''12'''||'''13'''||'''14'''||'''15'''||'''16'''||'''17'''
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|-
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|'''18'''||'''19'''||'''20'''||'''21'''||'''22'''||'''23'''||'''24'''
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|-
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|'''25'''||'''26'''||'''27'''||'''28'''||'''29'''||'''30'''||'''31'''
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|-
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| colspan="7"| <span style="color:#ffffff">-</span>
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|} <html></td>
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Revision as of 08:26, 22 September 2010

13/09/2010


Restriction digest of psiCHECK-2 plasmid
This will be used as backbone for raPCR cloning. Enzymes: XhoI and NotI

Assay:

5 µL 10x NEBuffer 3
5 µL 10x BSA
5 µL plasmid (psiCHECK-2, ~370 ng/µL)
3 µL XhoI
1 µL NotI
18.6 µL H2O

Restriction digest was performed for approx. 5h



raPCR to create binding sites for different miRNAs This random assembly PCR (raPCR) will be done to create binding site patterns for the miRNAs mentioned. In the first PCR step the oligos will basically anneal and constructs of different lengths will form. In the second step, the stop oligos are used as primers to amplify the previously formed constructs.

  • first tries are: hsa-mir-122, hsa-mir122(ran9-12) and mm-mir-376a/375 (Oligos: ra001-003 and ra006)

stop-oligos: raPCR_AS13-stop_rev_NotI and raPCR_AS13-stop_fw_XhoI (ra017/018)
spacer: raPCR_AS13-spacer(0) and raPCR_AS13-spacer(10) (ra012/013)

Oligos were used in standard conc. (100µM)

  • 1. PCR
Oligo mir-122 mir-122(ran9-12) mir-375/376a
miR 1 or 3 µL 1 or 3 µL 0.5 or 1.5 µL (each)
spacer(0)or (10) 1 µL 1 µL 1 µL
stop 0 or 0.5 µL each 0 or 0.5 µL each 0 or 0.5 µL each


Total: 12 reactions
each reaction was set up in 30 µL, using 2x Phusion Mastermix for 12 cycles

Temp Time
95°05:00
95°00:30x 12 cycles
57°00:45
72°00:45
4° forever
  • PCR purification: each PCR was purified using Qiagen PCR purification Kit and eluted in 32 µL

for the next PCR, three assay will tried:

  1. 5µL eluate + 1 µL of each stop oligo in 50µL total volume
  2. 5µL eluate + 2 µL of each stop oligo in 50µL total volume
  3. 20µL eluate + 1 µL of each stop oligo in 50µL total volume

stop-oligos: raPCR_AS13-stop_rev_NotI and raPCR_AS13-stop_fw_XhoI

  • 2. PCR

In total there were 72 reactions. Each was run with 2x Phusion Mastermix, missing volume was filled with water.

Temp Time
95°05:00
95°00:30x 25 cycles
65°00:45
72°00:50
72°05:00
4° forever

DNA was stored in fridge afterwards






July
MTWTFSS
1234
567891011
12131415161718
19202122232425
262728293031
-


August
MTWTFSS
1
2345678
9101112131415
16171819202122
23242526272829
3031


September
MTWTFSS
12345
6789101112
13141516171819
20212223242526
27282930
-


October
MTWTFSS
123
45678910
11121314151617
18192021222324
25262728293031
-
Retrieved from "http://2010.igem.org/User:Kleinsorg/test"