Haynes Lab:Notebook/CRISPR Editing/2014/09/15: Difference between revisions

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Ran 6 ul on gel.
Ran 6 ul on gel.<br>
[[Image:14.09.15 SRVYR control gel.png|400px]]
[[Image:14.09.15 SRVYR control gel.png|400px]]
<br>
<br>
Gel
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Band size'''
|-
| 1||Purified 'gRNA 20 +dox rep 1' PCR reaction with P197/P198||1660
|-
| 2||SURVEYOR Ctrl C PCR reaction||
|-
| 3||SURVEYOR Ctrl G PCR reaction||
|}
''After looking at the concentration of the sample DNA (3.212 with 260/280 of 3.4), I'd expect to not see anything on the gel but I see a decent band (lane 1)''
<br>
<br>
Top
Measured the concentration again, not sure what to make of this
:Purified 'gRNA 20 +dox rep 1' PCR reaction with P197/P198, SURVEYOR Ctrl C PCR reaction, and SURVEYOR Ctrl G PCR reaction.
 
Bottom
{|
:Luc->Cyan PCR reactions
| align="center" style="background:#f0f0f0;"|'''Sample Read#'''
::pure 1, 2a, 2b, pure 3a, pure 3b, 4a, 4b, pure 4c, 4c3
| align="center" style="background:#f0f0f0;"|'''260'''
| align="center" style="background:#f0f0f0;"|'''280'''
| align="center" style="background:#f0f0f0;"|'''260/280'''
| align="center" style="background:#f0f0f0;"|'''ng/µL'''
|-
| Nothing||0.005||0.003||1.643||4.841
|-
| gRNA 20 PCR||0.008||0.004||2.222||8.29
|-
| gRNA 20 PCR||0.014||0.007||1.928||13.619
|}
 
<br><br>
 
'Ran gel of all the Luc->AmCyan donor PCR reactions'


[[Image:14.09.15 PCR of Luc to AmCyan gel.png|600px]]
[[Image:14.09.15 PCR of Luc to AmCyan gel.png|600px]]
<br>
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Band size'''
|-
| 1||pure 1||729
|-
| 2*||2a||764
|-
| 3||2b||789
|-
| 4||pure 3a||834
|-
| 5||pure 3b||877
|-
| 6||4a||962
|-
| 7||4b||962
|-
| 8||pure 4c||918
|-
| 9||4c3||918
|}
*''in oww notebook, I wrote that I used P185, which shouldn't amplify this because it should amplify mCh. This length is assuming I used P186''
<br>
Looking back at the PCR I ran for the fourth round, the primers don't overlap with the templates so it makes sense that I didn't get the amplicons I was expecting. I am re-running round four with primers that actually overlap. <br>
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Template'''
| align="center" style="background:#f0f0f0;"|'''F primer'''
| align="center" style="background:#f0f0f0;"|'''R primer'''
| align="center" style="background:#f0f0f0;"|'''At'''
| align="center" style="background:#f0f0f0;"|'''length'''
|-
| 4e||3a (pure)||P178||P181||65||877
|}
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__NOTOC__
__NOTOC__

Revision as of 16:32, 15 September 2014

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09/15/2014

Purified 'gRNA 20 +dox rep 1' PCR reaction with P197/P198, SURVEYOR Ctrl C PCR reaction, and SURVEYOR Ctrl G PCR reaction. Ran binding through twice and water elution through twice. Eluted in 30ul.

Sample Read# 260 280 260/280 ng/µL
gRNA 20 +dox rep 1' PCR reaction with P197/P198 0.003 0.001 3.444 3.212
SURVEYOR Ctrl C PCR reaction 0.066 0.035 1.9 65.558
SURVEYOR Ctrl G PCR reaction 0.053 0.028 1.877 53.041


Ran 6 ul on gel.

Lane Sample Band size
1 Purified 'gRNA 20 +dox rep 1' PCR reaction with P197/P198 1660
2 SURVEYOR Ctrl C PCR reaction
3 SURVEYOR Ctrl G PCR reaction

After looking at the concentration of the sample DNA (3.212 with 260/280 of 3.4), I'd expect to not see anything on the gel but I see a decent band (lane 1)
Measured the concentration again, not sure what to make of this

Sample Read# 260 280 260/280 ng/µL
Nothing 0.005 0.003 1.643 4.841
gRNA 20 PCR 0.008 0.004 2.222 8.29
gRNA 20 PCR 0.014 0.007 1.928 13.619



'Ran gel of all the Luc->AmCyan donor PCR reactions'


Lane Sample Band size
1 pure 1 729
2* 2a 764
3 2b 789
4 pure 3a 834
5 pure 3b 877
6 4a 962
7 4b 962
8 pure 4c 918
9 4c3 918
  • in oww notebook, I wrote that I used P185, which shouldn't amplify this because it should amplify mCh. This length is assuming I used P186


Looking back at the PCR I ran for the fourth round, the primers don't overlap with the templates so it makes sense that I didn't get the amplicons I was expecting. I am re-running round four with primers that actually overlap.

Sample Template F primer R primer At length
4e 3a (pure) P178 P181 65 877



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