User:Karmella Haynes/Notebook/BioBrick cloning/2015/06/22: Difference between revisions

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CONCLUSIONS
CONCLUSIONS
* tba
* tba






----
----
'''Line item'''
'''Cas-tone - Phusion PCR for H3'''
 
* Minipreps
** Sigma GenElute kit, elute w/ 75 μL elution sln.
 
* Digests
** Check with #/# digests
 
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| rowspan="7" | <!-- [[Image:GelImage.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
|-
| DNA(plasmid) || 2.0 μL
|-
| 10X buffer || 1.5
|-
| EcoRI || 1.0
|-
| PstI || 1.0
|-
| dH<sub>2</sub>O || 9.5
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
|}
 
----
'''Line item'''
 
 
* Assemblies
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
 
 
* Digests (Fermentas FD)
** Specific notes
 
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| 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] -->
|-
| DNA (plasmid) || up to 25 μL
|-
| 10x buffer || 3.0
|-
| enzyme 1 || 1.0
|-
| enzyme 2 || 1.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
|}


* Phusion PCR-amplify ORFs
# mEmerald-H3-23, H3_1 f1 / H3_40 r1
# mEmerald-H3-23, H3_1 f1 / H3_60 r1
# mEmerald-H3-23, H3_1 f1 / H3_80 r1
# mEmerald-H3-23, H3_1 f1 / H3_116 r1
# mEmerald-H3-23, H3_1 f1 / H3_136 r1


* Measure conc.'s
{| {{table}} cellspacing="3" <!-- PCR rxn table -->
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf
| Sample || OD260 || 260/280 || ng/μL
|-
| 1. Digested part (a/b) || --- || --- || ---
|-
| 2. Digested part (c/d) || --- || --- || ---
|}
 
 
* Dephosphorylation (Roche)
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Vol
|-
| bgcolor=#cfcfcf | Mix (x6)
| DNA (clean digest) || up to 17 μL (500 ng)
| rowspan=7 | Expected:<br>1. H3-1-40 = 129<br>2. H3-1-60 = 189<br>3. H3-1-80 = 250<br>4. H3-1-116 = 373<br>5. H3-1-136 = 433
|-
| rowspan=7 | [[Image:KAH052015_gel1.jpg|250px|Hover name]]<br>5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| 10x buffer d.p. || 2.0
|-
| phosphatase || 1.0
|-
|-
| dH<sub>2</sub>O || ---
| Plasmid DNA || 0.2 || 1.2
|-
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
| 10 μM H3_1 f1 || 1.0  || 6.0 
|}
 
 
* Ligations
# insert/(a/b)/size + vector/(c/d)/size
# vector/(c/d)/size
 
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn1
| bgcolor=#cfcfcf | Rxn2
|-
|-
| Insert DNA        || ### || ---  
| 10 μM r primer || 1.0 || ---
|-
|-
| Vector DNA        || ### || ###
| 10 mM dNTPs || 1.0 || 6.0
|-
|-
| 2x lgn buf (Roche) || ###  || ###
| 5x HF buffer  || 10.0 || 60.0
|-
|-
| T4 ligase (NEB)    || 1.0  || 1.0  
| Phusion Pol.  || 0.5 || 3.0  
|-
|-
| dH<sub>2</sub>O   || ### || ###
| dH<sub>2</sub>O || 36.3 || 217.8
|-
|-
| &nbsp;             || # μL || # μL
| &nbsp;       || 50.0 μL ||
|}
|}


Thermal cycler: Bio-Rad - Phusion
* 98°C 3 min.
* 30x[98°C, 10 sec; 70°C 30 sec; 72°C 30 sec]
* 72°C 3 min.
* 4°C, ∞




Revision as of 16:42, 22 June 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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06/22/15

  • Rene - CRISPR PCR library PCR
  • Cas-tone - PCR new inserts


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 = unique clone
      • A01, A02, etc. = well position in dish or spot on agar array

1-96. L34_001_A01 - L34_096_H12 (full plate)
97-192. G34_001_A01 - G34_096_H12 (full plate)


Reagent Rxn1-24 Mix (x104) Expected:
PCR insert/ pJET = ~600 bp 		Hover name
10 μL/lane, 1% agarose; Ladder
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"

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


CONCLUSIONS

  • tba


Cas-tone - Phusion PCR for H3

  • Phusion PCR-amplify ORFs
  1. mEmerald-H3-23, H3_1 f1 / H3_40 r1
  2. mEmerald-H3-23, H3_1 f1 / H3_60 r1
  3. mEmerald-H3-23, H3_1 f1 / H3_80 r1
  4. mEmerald-H3-23, H3_1 f1 / H3_116 r1
  5. mEmerald-H3-23, H3_1 f1 / H3_136 r1
Reagent Vol Mix (x6) Expected:
1. H3-1-40 = 129
2. H3-1-60 = 189
3. H3-1-80 = 250
4. H3-1-116 = 373
5. H3-1-136 = 433 		Hover name
5 μL/lane; 1% agarose; Ladder
Plasmid DNA 0.2 1.2
10 μM H3_1 f1 1.0 6.0
10 μM r primer 1.0 ---
10 mM dNTPs 1.0 6.0
5x HF buffer 10.0 60.0
Phusion Pol. 0.5 3.0
dH2O 36.3 217.8
  50.0 μL

Thermal cycler: Bio-Rad - Phusion

  • 98°C 3 min.
  • 30x[98°C, 10 sec; 70°C 30 sec; 72°C 30 sec]
  • 72°C 3 min.
  • 4°C, ∞



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