Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/21: Difference between revisions
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==Restriction Digest== | ==Restriction Digest== | ||
In two separate tubes, digest the DBN001 (gBlock) and the vector using the following reagents. Use plenty of DNA for this step; the objective is to have an abundance of digested material to work with downstream. | In two separate tubes, digest the DBN001 insert (gBlock) and the mCh_pSB1A3 vector using the following reagents. Use plenty of DNA for this step; the objective is to have an abundance of digested material to work with downstream. | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Reagent''' | | align="center" style="background:#f0f0f0;"|'''Reagent''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''Insert (µL)''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''Vector (µL)''' | ||
|- | |- | ||
| DNA||10 µL||20 | | DNA||10 µL||20 | ||
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| 10x FastDigest Buffer||3||3 | | 10x FastDigest Buffer||3||3 |
Revision as of 10:39, 22 April 2015
Cloning DBN001 into pSB1A3 | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||||||||||||||||||||||||||||||||||
OverviewTrying the ligation again (previous attempt), with major revisions as suggested by Dr. Haynes. DBN001 is the gBlock containing homology arms for insertion into Gal4-EED/Luc and Type IIS cloning sites to add HPK-CFP. pSB1A3 is a standard cloning vector for molecular biology work. The plasmid I'm using contains an mCherry insert, so I can easily recognize colonies containing the DBN001 insert by their lack of red coloration. Remember to use one negative control (water), one negative control (digested backbone), and one positive control (untreated vector). Major steps:
mCherry vector concentration is 392 ng/µL. gBlock concentration is 311 ng/µL. Restriction DigestIn two separate tubes, digest the DBN001 insert (gBlock) and the mCh_pSB1A3 vector using the following reagents. Use plenty of DNA for this step; the objective is to have an abundance of digested material to work with downstream.
Mix and incubate at 37°C for 15 minutes. Skip heat inactivation (you'll be purifying the restriction digest products). 'PCR' Clean-up of digested DBN001Follow the instructions in the Sigma PCR clean-up kit. Gel purification of pSB1A3 backboneLoad entire volume of digest into one lane of the gel. Gel shows three bands: one at approx. 1000bp, one at approx 2000bp and one at over 3000bp (brighter). 3000bp fragment is probably incomplete digestion of plasmid, either single cut or uncut. Excised 2000bp band (backbone without insert) and proceeded to gel purification. Mass of band fragment excised from gel: 0.254mg Follow the instructions in the Sigma gel extraction kit. DNA Quantification
LigationLigate using Roche DNA Ligase buffer and NEB T4 Ligase. To determine amount of insert to use in the ligation reaction:
Round up to 15ng to be on the safe side.
Incubate at room temp. for 10 minutes, then heat inactivate at 65°C for 10 minutes. TransformationTransform cells with entire volume of ligated plasmid. Remember to use negative and positive controls (water and untreated vector, respectively).
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