User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/16: Difference between revisions
From OpenWetWare
(fix raw html notebook nav) |
|||
(23 intermediate revisions by one other user not shown) | |||
Line 2: | Line 2: | ||
|- | |- | ||
|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}}}}]]}} | ||
|- | |- | ||
| colspan="2"| | | colspan="2"| | ||
Line 8: | Line 8: | ||
==04/16/15== | ==04/16/15== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* LCR Development - PCR MV10 | * LCR Development - PCR MV10 backbone | ||
* gRNA cloning (Rene) | |||
Line 20: | Line 20: | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
|- | |- | ||
| DNA(plasmid) || 5.0 μL | | DNA(plasmid) || 5.0 μL | ||
Line 45: | Line 43: | ||
| 1. MV10 XbaI || 0.017 || 1.47 || 17.24 | | 1. MV10 XbaI || 0.017 || 1.47 || 17.24 | ||
|} | |} | ||
Phusion PCR | |||
# 1.0 μL template, HF buffer | |||
# 2.0 μL template, HF buffer | |||
# 1.0 μL template, GC buffer | |||
# 2.0 μL template, GC buffer | |||
{| {{table}} cellspacing="3" <!-- PCR rxn. table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Rxn1,3 | |||
| bgcolor=#cfcfcf | Rxn2,4 | |||
| rowspan="7" | Expected:<br>MV10 band = 5191 | |||
| rowspan="7" | [[Image:KAH041615_gel1.jpg|250px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |||
| Template || 1.0 || 2.0 | |||
|- | |||
| 10 uM fwd primer || 1.0 || 1.0 | |||
|- | |||
| 10 uM rev primer || 1.0 || 1.0 | |||
|- | |||
| 10 mM dNTPs || 1.0 || 1.0 | |||
|- | |||
| Phusion pol. || 0.5 || 0.5 | |||
|- | |||
| 5x buffer (HF/GC) || 5.0 || 5.0 | |||
|- | |||
| dH<sub>2</sub>O || 40.5 || 39.5 | |||
|- | |||
| || 50.0 || 50.0 | |||
|} | |||
<font color=red>Mistake: Buffer volume should be 10/rxn, no 5! Consider re-run</font> | |||
Program: Phusion (block B) | |||
* 98°C, 3 min | |||
* 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min] | |||
* 72°C, 10 min | |||
* 4°C ∞ | |||
Conclusion | |||
* Not ideal, proceed with caution | |||
Measure conc. | |||
* PCR 1 & 2 combined, purified with Sigma PCR clean-up kit | |||
* Eluted& back-eluted with 30 μL elution sln. | |||
{| {{table}} cellspacing="3" <!-- [DNA] table --> | |||
|- bgcolor=#cfcfcf | |||
| Sample || OD260 || 260/280 || ng/μL | |||
|- | |||
| 1. 5'p-MV10 PCR || 0.008 || 1.28 || 7.77 | |||
|} | |||
---- | |||
'''gRNA cloning - Rene''' | |||
* Follow the "Oligo annealing and cloning into backbone vectors - NEW" protocol | |||
Phosphorylate and anneal oligo pairs | |||
# g051 T/B | |||
# g052 T/B | |||
# g053 T/B | |||
# g054 T/B | |||
# g055 T/B | |||
# g056 T/B | |||
{| {{table}} cellspacing="3" <!-- Oligo annealing table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Rxn | |||
| bgcolor=#cfcfcf | Mix (7x) | |||
|- | |||
| 100 μM Oligo 1 || 1.0 || --- | |||
|- | |||
| 100 μM Oligo 2 || 1.0 || --- | |||
|- | |||
| 10x T4 Lign buf (NEB) || 1.0 || 7.0 | |||
|- | |||
| T4 PNK (NEB) || 0.5 || 3.5 | |||
|- | |||
| dH<sub>2</sub>O || 6.5 || 45.5 | |||
|- | |||
| || 10.0 | |||
|} | |||
LabNet OptiMax Thermocycler: AnOlig RD | |||
* 37°C, 30 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, ∞ | |||
Dilute the product(s) 1:250 | |||
* Add 2 μL product to 498 μL dH<sub>2</sub>O | |||
Digestion/ Ligation Reactions (Vector / dsOligo Insert) | |||
# pX330 / g051 | |||
# pX330 / g052 | |||
# pX330 / g053 | |||
# pX330 / g054 | |||
# pX330 / g055 | |||
# pX330 / g056 | |||
{| {{table}} cellspacing="3" <!-- Oligo annealing table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Rxn | |||
| bgcolor=#cfcfcf | Mix (x7) | |||
|- | |||
| 100 ng Vector || 0.5 || 3.5 | |||
|- | |||
| 1:250 dsOligo || 2.0 || --- | |||
|- | |||
| 10x FD buf || 2.0 || 14.0 | |||
|- | |||
| 10 mM DTT || 1.0 || 7.0 | |||
|- | |||
| 10 mM ATP || 1.0 || 7.0 | |||
|- | |||
| FastDigest BbsI || 1.0 || 7.0 | |||
|- | |||
| T4 DNA ligase || 0.5 || 3.5 | |||
|- | |||
| dH<sub>2</sub>O || 12.0 || 84.0 | |||
|- | |||
| || 20.0 | |||
|} | |||
LabNet OptiMax Thermocycler: BbsI Dig/Lig | |||
* 6x [37°C, 5 min; 23°C, 5 min] | |||
* 4°C, ∞ | |||
Transformation(s) | |||
* Split ligations in 1/2, do rapid protocol for 1 set, long protocol for other set if needed | |||
* 10.0 μL ligation + 50 μL DH5α-turbo | |||
* Plate on 100 μg/mL amp | |||
RESULTS (4/17/15) | |||
* Success! Plates 1-3, 5, 6 had ~5 colonies. Plate 4 had just one. | |||
* Pick two colonies from each (except plate 4, pick 1) | |||
* Make streak plate & set up 5 mL cultures | |||
Latest revision as of 00:54, 27 September 2017
Karmella's BioBrick Cloning | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
04/16/15
LCR Development
Measure conc.
Mistake: Buffer volume should be 10/rxn, no 5! Consider re-run Program: Phusion (block B)
Conclusion
gRNA cloning - Rene
Phosphorylate and anneal oligo pairs
LabNet OptiMax Thermocycler: BbsI Dig/Lig
RESULTS (4/17/15)
|