User:Karmella Haynes/Notebook/BioBrick cloning/2015/06/23: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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==mm/dd/yy==
==06/23/15==
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* Line item 1
* Cas-tone - Assemblies: H3 parts into MV11
* Line item 2
* Rene - CRISPR library, continue dirty PCR




----
----
'''Assemblies'''
'''Cas-tone Assemblies'''
* Do-over - use inserts with improved reverse primer (eliminate frame-shift)
# H3-1-40_MV11: H3-1-40/(X/N)/129 + MV11/(S/N)/9506
# H3-1-40_MV11: H3-1-40/(X/N)/129 + MV11/(S/N)/9506
# H3-1-60_MV11: H3-1-60/(X/N)/189 + "
# H3-1-60_MV11: H3-1-60/(X/N)/189 + "
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* Digest of vector (Fermentas FD)
* Digest of vector (Fermentas FD)
** SpeI/NotI
# MV11 - SpeI/NotI


{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
Line 28: Line 29:
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <!-- [[Image:GelImage.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
| rowspan="7" | [[Image:KAH062315_gel1.jpg|150px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA (plasmid) || 15.0
| DNA (plasmid) || 20.0
|-
|-
| 10x buffer || 3.0
| 10x buffer || 3.0
Line 38: Line 39:
| NotI || 1.0
| NotI || 1.0
|-
|-
| dH<sub>2</sub>O || 10.0
| dH<sub>2</sub>O || 5.0
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~15 min.
| &nbsp; || 30 μL --> 37°C/ ~15 min.
Line 48: Line 49:




* DpnI digest & clean-up of PCR products
* DpnI digest & clean-up of PCR products (inserts)
** Add 1 μL DpnI to each PCR rxn. Incubate @ 37°C/ 5 min.
** Add 1 μL DpnI to each PCR rxn. Incubate @ 37°C/ 5 min.
** Use the Qiagen Qiaquick PCR Purification kit (cat. no. 28104 ) to purify DNA.
** Use the Qiagen Qiaquick PCR Purification kit (cat. no. 28104 ) to purify DNA.
Line 59: Line 60:
| Sample || OD260 || 260/280 || ng/μL
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. MV11 (S/N) || 0.03 || 1.879 || 13.5
| 1. MV11 (S/N) || 0.019 || 1.596 || 19.4
|-
|-
| 2. H3-1-40 PCR || 0.03 || 1.879 || 29.8
| 2. H3-1-40 PCR || 0.024 || 1.908 || 24.2
|-
|-
| 3. H3-1-60 PCR || 0.04 || 1.974 || 39.7
| 3. H3-1-60 PCR || 0.041 || 1.984 || 40.8
|-
|-
| 4. H3-1-80 PCR || 0.047 || 1.865 || 46.8
| 4. H3-1-80 PCR || 0.044 || 1.917 || 43.8
|-
|-
| 5. H3-1-116 PCR || 0.06 || 1.887 || 59.57
| 5. H3-1-116 PCR || 0.067 || 1.865 || 67.1
|-
|-
| 6. H3-1-136 PCR || 0.062 || 1.932 || 62.0
| 6. H3-1-136 PCR || 0.072 || 1.901 || 72.4
|}
|}




* Digest & dephos inserts
* Digest & dephos inserts
** XbaI/NotI


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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| 10X FD buffer || 2.0
| 10X FD buffer || 2.0
|-
|-
| FD EcoRI || 1.0
| FD XbaI || 1.0
|-
|-
| FD XbaI || 1.0  
| FD NotI || 1.0  
|-
|-
| Roche SAP || 1.0
| Roche SAP || 1.0
Line 118: Line 120:
| Insert DNA        || 0.5  || ---   
| Insert DNA        || 0.5  || ---   
|-
|-
| Vector DNA        || 3.5  || 3.5   
| Vector DNA        || 2.5  || 2.5   
|-
|-
| 2x lgn buf (Roche) || 5.0  || 5.0  
| 2x lgn buf (Roche) || 5.0  || 5.0  
Line 124: Line 126:
| T4 ligase (NEB)    || 1.0  || 1.0   
| T4 ligase (NEB)    || 1.0  || 1.0   
|-
|-
| dH<sub>2</sub>O    || --- || 0.5  
| dH<sub>2</sub>O    || 1.0 || 1.5  
|-
|-
| &nbsp;            || 10.0 μL || 10.0 μL  
| &nbsp;            || 10.0 μL || 10.0 μL  
|}
|}


RESULTS (5/22/15)
RESULTS (6/24/15)
* Success! Ligation plates have 15-20 colonies, neg. ctrl has 1 colony
* SUCCESS! Plate 6 (neg ctrl) has 7 colonies; Plates 1-5 have 30 - 50 colonies
* Pick 2 colonies from each plate for 5 mL cultures and streaks
* Pick two clones from each for 5 mL cultures/ minipreps
 
 
 
----
 
'''Rene - CRISPR PCR library PCR'''
* '''Part 1 - GoTaq "Dirty" PCR on liquid mini-cultures, 600 bp amplicons; primers P163/P215'''
* Part 2 - Nested SYBR real time PCR & melt curves on (1) mini cultures, (2) amplicons from GoTaq
 
 
* Part 1 - GoTaq "Dirty" PCR on liquid mini-cultures, 600 bp amplicons
** Clone labeling notes, changed from last time:
*** L34 = Luc14 cells treated with gRNA034
*** G34 = Gal4EED/luc cells treated with gRNA034
*** 001, 002, 003 = clone no.
*** A01, A02, etc. = well position in dish or spot on agar array
 
1-96. L34_097_A01 - L34_192_H12 (full plate)<br>
 
 
{| {{table}} cellspacing="3" <!-- PCR rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn1-24
| bgcolor=#cfcfcf | Mix (x104)
| rowspan="7" | Expected:<br>PCR insert/ pJET = ~600 bp<br>1. L34_097_A01<br>2. L34_109_B01<br>3. L34_121_C01<br>4. L34_133_D01<br>5. L34_145_E01<br>6. L34_157_F01<br>7. L34_169_G01<br>8. L34_181_H01
| rowspan="7" | Gel showed no signal
|-
| Template (culture) || 2.0 || ---
|-
| 10 uM fwd primer || 1.0 || 104.0
|-
| 10 uM rev primer || 1.0 || 104.0
|-
| 2x GoTaq (clear) || 12.5 || 1300.0
|-
| dH<sub>2</sub>O || 8.5 || 884.0
|-
| &nbsp; || 25.0 μL
|}
 
* Note: Master Mix Multiplier (for one 96-well plate) = 96 + 1 extra 8-well column = 104
* Aliquot 200 μL into 8 tubes in 8-tube strip
* Use multichannel to aliquot 23 μL to 96-well plate, 1 column at a time
* Refill the 8-tube strip as needed with enough to fill remaining columns
 
 
PCR - Labnet: "GoTaq35KAH"
* '''98°C, 10 min'''
* 35x[95°C, 10 sec; 60°C, 10 sec; 72°C, 20 sec]
* 72°C, 3 min
* 4°C ∞
 
 
CONCLUSIONS
* Looks like amplification failed
* Move on to sequencing of all clones
* Restart fresh cultures tomorrow
* Will submit as pellets to DNASU




Latest revision as of 01:01, 27 September 2017

Karmella's BioBrick Cloning Main project page
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06/23/15

  • Cas-tone - Assemblies: H3 parts into MV11
  • Rene - CRISPR library, continue dirty PCR


Cas-tone Assemblies

  • Do-over - use inserts with improved reverse primer (eliminate frame-shift)
  1. H3-1-40_MV11: H3-1-40/(X/N)/129 + MV11/(S/N)/9506
  2. H3-1-60_MV11: H3-1-60/(X/N)/189 + "
  3. H3-1-80_MV11: H3-1-80/(X/N)/250 + "
  4. H3-1-116_MV11: H3-1-116/(X/N)/373 + "
  5. H3-1-136_MV11: H3-1-136/(X/N)/433 + "


  • Digest of vector (Fermentas FD)
  1. MV11 - SpeI/NotI
Reagent Volume Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 20.0
10x buffer 3.0
SpeI 1.0
NotI 1.0
dH2O 5.0
  30 μL --> 37°C/ ~15 min.
  • Gel purification
    • Sigma GenElute kit. Elute & back elute w/ 25 μL elution sln.


  • DpnI digest & clean-up of PCR products (inserts)
    • Add 1 μL DpnI to each PCR rxn. Incubate @ 37°C/ 5 min.
    • Use the Qiagen Qiaquick PCR Purification kit (cat. no. 28104 ) to purify DNA.
    • Elute & back-elute with 30 μL of elution solution.


  • Measure [DNA] for vector and inserts
Sample OD260 260/280 ng/μL
1. MV11 (S/N) 0.019 1.596 19.4
2. H3-1-40 PCR 0.024 1.908 24.2
3. H3-1-60 PCR 0.041 1.984 40.8
4. H3-1-80 PCR 0.044 1.917 43.8
5. H3-1-116 PCR 0.067 1.865 67.1
6. H3-1-136 PCR 0.072 1.901 72.4


  • Digest & dephos inserts
    • XbaI/NotI
Reagent Volume
DNA (500 ng) up to 15.0 μL
10X FD buffer 2.0
FD XbaI 1.0
FD NotI 1.0
Roche SAP 1.0
dH2O x μL
  20.0

Thermal cycler: LabNet OptiMax "AnOlig shrt"

  • 37°C, 10 min
  • 95°C, 5 min
  • Ramp down to 25°C, 5°C/1 min. [90/1 min, 85/1 min, 80/1 min, ... 25°C/1 min]
  • 25°C, ∞
  • Final concentration = 25 ng/μL.


  • Ligations
    • > 2:1 ratio calculations...
    • 433 bp largest insert / 9506 bp vector * 2 * 50 ng vector = 4.5 ng
    • 129 bp smallest insert / 9506 bp vector * 2 * 50 ng vector = 1.4 ng
Reagent Rxn1-5 Rxn6
Insert DNA 0.5 ---
Vector DNA 2.5 2.5
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 1.0 1.5
  10.0 μL 10.0 μL

RESULTS (6/24/15)

  • SUCCESS! Plate 6 (neg ctrl) has 7 colonies; Plates 1-5 have 30 - 50 colonies
  • Pick two clones from each for 5 mL cultures/ minipreps


Rene - CRISPR PCR library PCR

  • Part 1 - GoTaq "Dirty" PCR on liquid mini-cultures, 600 bp amplicons; primers P163/P215
  • Part 2 - Nested SYBR real time PCR & melt curves on (1) mini cultures, (2) amplicons from GoTaq


  • Part 1 - GoTaq "Dirty" PCR on liquid mini-cultures, 600 bp amplicons
    • Clone labeling notes, changed from last time:
      • L34 = Luc14 cells treated with gRNA034
      • G34 = Gal4EED/luc cells treated with gRNA034
      • 001, 002, 003 = clone no.
      • A01, A02, etc. = well position in dish or spot on agar array

1-96. L34_097_A01 - L34_192_H12 (full plate)


Reagent Rxn1-24 Mix (x104) Expected:
PCR insert/ pJET = ~600 bp
1. L34_097_A01
2. L34_109_B01
3. L34_121_C01
4. L34_133_D01
5. L34_145_E01
6. L34_157_F01
7. L34_169_G01
8. L34_181_H01 		Gel showed no signal
Template (culture) 2.0 ---
10 uM fwd primer 1.0 104.0
10 uM rev primer 1.0 104.0
2x GoTaq (clear) 12.5 1300.0
dH2O 8.5 884.0
  25.0 μL
  • Note: Master Mix Multiplier (for one 96-well plate) = 96 + 1 extra 8-well column = 104
  • Aliquot 200 μL into 8 tubes in 8-tube strip
  • Use multichannel to aliquot 23 μL to 96-well plate, 1 column at a time
  • Refill the 8-tube strip as needed with enough to fill remaining columns


PCR - Labnet: "GoTaq35KAH"

  • 98°C, 10 min
  • 35x[95°C, 10 sec; 60°C, 10 sec; 72°C, 20 sec]
  • 72°C, 3 min
  • 4°C ∞


CONCLUSIONS

  • Looks like amplification failed
  • Move on to sequencing of all clones
  • Restart fresh cultures tomorrow
  • Will submit as pellets to DNASU



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