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"| | |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]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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| colspan="2"| | | colspan="2"| | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
== | ==06/23/15== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* | * 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) | |||
# H3-1-40_MV11: H3-1-40/(X/N)/129 + MV11/(S/N)/9506 | |||
# H3-1-60_MV11: H3-1-60/(X/N)/189 + " | |||
# H3-1-80_MV11: H3-1-80/(X/N)/250 + " | |||
# H3-1-116_MV11: H3-1-116/(X/N)/373 + " | |||
# H3-1-136_MV11: H3-1-136/(X/N)/433 + " | |||
* | * Digest of vector (Fermentas FD) | ||
# MV11 - SpeI/NotI | |||
{| {{table}} | {| {{table}} cellspacing="3" <!-- Digest rxn. table --> | ||
|- valign="top" | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | | | 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) || | | DNA (plasmid) || 20.0 | ||
|- | |- | ||
| | | 10x buffer || 3.0 | ||
|- | |- | ||
| | | SpeI || 1.0 | ||
|- | |- | ||
| | | NotI || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 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 | ||
# | {| {{table}} cellspacing="3" <!-- [DNA] table --> | ||
|- bgcolor=#cfcfcf | |||
| 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 | ||
{| {{table}} cellspacing="3" <!-- Digest rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
|- valign="top" | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
|- | |- | ||
| DNA ( | | 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 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 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 | |||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | |||
{| {{table}} cellspacing="3" <!-- | |- bgcolor="#cfcfcf" valign="top" | ||
| Reagent | |||
| Rxn1-5 | |||
| Rxn6 | |||
|- | |- | ||
| | | Insert DNA || 0.5 || --- | ||
| | |||
|- | |- | ||
| DNA | | Vector DNA || 2.5 || 2.5 | ||
|- | |- | ||
| | | 2x lgn buf (Roche) || 5.0 || 5.0 | ||
|- | |- | ||
| | | T4 ligase (NEB) || 1.0 || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 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)<br> | |||
{| {{table}} cellspacing="3" <!-- | {| {{table}} cellspacing="3" <!-- PCR rxn. table --> | ||
|- valign="top" | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Rxn1 | | bgcolor=#cfcfcf | Rxn1-24 | ||
| bgcolor=#cfcfcf | | | 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 | | dH<sub>2</sub>O || 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 | |||
Latest revision as of 01:01, 27 September 2017
Karmella's BioBrick Cloning | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
06/23/15
Cas-tone Assemblies
Thermal cycler: LabNet OptiMax "AnOlig shrt"
RESULTS (6/24/15)
Rene - CRISPR PCR library PCR
1-96. L34_097_A01 - L34_192_H12 (full plate)
|