User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/27: 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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** Total wells: 36 (1 well per rxn.)
** Total wells: 36 (1 well per rxn.)
** '''Template+water Multiplier''' = none (use 10x diluted stock for all rxns)
** '''Template+water Multiplier''' = none (use 10x diluted stock for all rxns)
** '''Primer+SYBR Mix Multiplier''' = 36 + 3 = '''39'''
** '''Primer+SYBR Mix Multiplier''' = 27 + 3 = '''39'''




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# Lu34_AA01 - 12
# Lu34_AA01 - 12
# Ga34_AA01 - 12
# Ga34_AA01 - 12
# nTc
# nTc (3 wells)




* Master Mix: Primers+SYBR
* Master Mix: Primers+SYBR
{|
** F = P149
** R = P160
** 10.5 per well
{| {{table}}
|-
|-
|  Reagent || Volume || (x39)
|  Reagent || Volume || (x30)
|-
|-
| 750 nM F/R primers || 3.0 || 117.0
| 750 nM F/R primers || 3.0 || 90.0
|-
|-
| 2x SYBR MM || 7.5 || 292.5
| 2x SYBR MM || 7.5 || 225
|-
|-
| &nbsp; || 15.0
| &nbsp; || 15.0
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* Run PCR - Roche LC480
* Plate loading
** 95°C, 3 min
** Pipette 21 Primers+SYBR (2 x 10.5) into first row (A1-12)
** 30x [95°C, 10 sec / 57°C, 10 sec / 72°C, 10 sec (measure)]
** Multichannel-aliquot 10.5 Primers+SYBR into next row (B1-12)
** 72°C, 30 sec
** Pipette 10.5 Primers+SYBR into C1-3 (nTc wells)
** Melt: 70°C -- +0.1°C/ 5 sec (measure) --> 95°C
** Multi-channel pipette 4.5 Template+water into appropriate wells (A1-12, B1-12)
** Pipette 4.5 water into C1-3 (nTc wells)




* Run PCR - Roche LC480: "SYBR Karmella CNV melt curve Template"
** 95°C, 5 min
** 30x [95°C, 10 sec; 60°C, 10 sec; 72°C, 10 sec (measure)]
** Melt: continuous acquisition; 95°C, 4 sec; 70°C, 60 sec, -- +0.1°C/ 5 sec (programmed as "6 acquisitions/°C") --> 95°C
** Cooling: 40°C, 30 sec






RESULTS:
# Lu34_AA01 - 12 -- Melt temps 85.1 ± 0.1 °C; additional short peak ~87°C
# Ga34_AA01 - 12 -- Melt temps 85.1 ± 0.1 °C; additional short peak ~87°C
# nTc (3 wells) -- no amplification


* Try using more diluted template (100x)
* Continue by sequencing a few purified PCR products with P215 to find a reference sample (wild type sequence)


* 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.
|}
* Measure conc.'s
{| {{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 | Volume
|-
| DNA (clean digest) || up to 17 μL (500 ng)
|-
| 10x buffer d.p. || 2.0
|-
| phosphatase || 1.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|}
* 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        || ###  || ---
|-
| Vector DNA        || ###  || ###
|-
| 2x lgn buf (Roche) || ###  || ###
|-
| T4 ligase (NEB)    || 1.0  || 1.0
|-
| dH<sub>2</sub>O    || ###  || ###
|-
| &nbsp;            || # μL || # μL
|}




Latest revision as of 00:59, 27 September 2017

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

05/27/15

  • Rene - hi-res melt curve screen on CRISPR clones
  • Ben - continue luc replacement donor cloning


Rene - hi-res melt curve screen on CRISPR clones


  • Set-up
    • 2 replicates per clone
    • Total wells: 36 (1 well per rxn.)
    • Template+water Multiplier = none (use 10x diluted stock for all rxns)
    • Primer+SYBR Mix Multiplier = 27 + 3 = 39


  • Template+ Water: Dilute PCR reaction 10x
    • Use multichannel to transfer 5.0 μL PCR products into 45.0 μL dH2O
    • Set up dilutions in 8-tube strips
    • Use 4.5 μL Template+water per rxn
  1. Lu34_AA01 - 12
  2. Ga34_AA01 - 12
  3. nTc (3 wells)


  • Master Mix: Primers+SYBR
    • F = P149
    • R = P160
    • 10.5 per well
Reagent Volume (x30)
750 nM F/R primers 3.0 90.0
2x SYBR MM 7.5 225
  15.0


  • Plate loading
    • Pipette 21 Primers+SYBR (2 x 10.5) into first row (A1-12)
    • Multichannel-aliquot 10.5 Primers+SYBR into next row (B1-12)
    • Pipette 10.5 Primers+SYBR into C1-3 (nTc wells)
    • Multi-channel pipette 4.5 Template+water into appropriate wells (A1-12, B1-12)
    • Pipette 4.5 water into C1-3 (nTc wells)


  • Run PCR - Roche LC480: "SYBR Karmella CNV melt curve Template"
    • 95°C, 5 min
    • 30x [95°C, 10 sec; 60°C, 10 sec; 72°C, 10 sec (measure)]
    • Melt: continuous acquisition; 95°C, 4 sec; 70°C, 60 sec, -- +0.1°C/ 5 sec (programmed as "6 acquisitions/°C") --> 95°C
    • Cooling: 40°C, 30 sec


RESULTS:

  1. Lu34_AA01 - 12 -- Melt temps 85.1 ± 0.1 °C; additional short peak ~87°C
  2. Ga34_AA01 - 12 -- Melt temps 85.1 ± 0.1 °C; additional short peak ~87°C
  3. nTc (3 wells) -- no amplification
  • Try using more diluted template (100x)
  • Continue by sequencing a few purified PCR products with P215 to find a reference sample (wild type sequence)




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