Links and notes on basic protocols:
Protocol sites and publications:
Cold Spring Harbor Protocols: http://cshprotocols.cshlp.org/
Mixing of dNTPs.
A 10 mM dNTP mix should contain 10 mM dNTPs total (2.5 mM of each). To prepare 40 uL 10 mM dNTP, mix:
1 uL 100 mM dATP 1 uL 100 mM dGTP 1 uL 100 mM dCTP 1 uL 100 mM dTTP 36 uL H2O.
~100 ng equivalents backbone DNA. 3:1 - 5:1 molar ratio insert/backbone 2 uL T4 DNA ligase buffer 0.5 uL T4 DNA ligase H2O to 20 uL.
Assuming a backbone concentration of ~20 ng/uL, an insert concentration of about 10 ng/uL, insert:backbone molar ratio = 3:1, and insert size 100 bp, this corresponds to roughly 5-7.5 uL backbone + 0.5 uL insert.
Incubate at 16 C for 3 h or overnight.
Transformation of E. coli:
Transform using 1 uL plasmid DNA added to 0.1 mL supercompetent cells. For ligation mixtures, use 2-5 uL. Incubate on ice for 30 min. Heat shock at 42 C for 45 s. Incubate 2 min on ice, then add 200 SOC or LB medium. After heat-shock and addition of medium, incubate 30 min - 1 h for Kan-resistant cells. Spread 100 uL per plate (maximum 200 uL per plate).
General purpose restriction digest of purified PCR product:
After purification of PCR product and elution with 50 uL EB, mix: 43 uL purified PCR product 5 uL 10 x restriction enzyme buffer 1 uL of each restriction enzyme.
Alternatively, add directly to the tube with purified PCR product (slightly less than 50 uL), add: 5.5 uL 10x restriction enzyme buffer 1 uL of each restriction enzyme. (Suggestion only. Check if this method is suitable for routine use.)
To achieve more reliable results, follow the below procedure:
Initialize instrument Blank with water Measure concentration of DNA in water (should be close to zero) Measure concentration of DNA in buffer. Note if measurement on buffer gives a reading (wrongly) indicating DNA presence when using water as blank. Blank against buffer. Measure DNA concentration in buffer (should be zero).
Measure first sample. Wipe pedestal clean. Perform measurement using buffer as sample. Repeat until reading is close to zero. Measure next sample.
Outline of a general strategy for a restriction-ligation cloning project:
Digest 1 ug backbone plasmid for 2 h in 20 uL, add 1 uL CIP and incubate for a further 1 h. Purify with PCR purification kit.
Perform PCR to amplify insert sequence with added restriction sites. Purify PCR product. Digest PCR product and purify again.
Set up a ligation mixture with backbone and insert. Always include a backbone-only ligation control and a uncut vector plasmid positive control. If the insert was amplified from a template with the same selection marker as the backbone, also include an insert-only control.
Consider also including a cut backbone only (not ligated) negative control.
Ligate for 3 h at 16 C. Remove 5 uL for transformation and continue incubation of the remaining amount overnight. Inspect plates next morning. If no colonies were obtained, repeat transformation using the ligation mixture incubated overnight.
If background of backbone-only ligation control is high, and it was not performed previously, re-consider performing a backbone-only no-ligation control to determine if the problem is re-ligation of vector plasmid, or uncut vector plasmid.
If colonies are obtained on vector-insert ligation plates, compare with negative control plates and pick a suitable number of colonies. Inoculate each colony into ~12 mL LB with antibiotic and incubate overnight.
For each inoculant, prepare a glycerol stock and proceed to miniprep with the remaining culture.
For low-copy plasmids, elute miniprep DNA in 50 uL H2O/buffer to achieve a higher concentration.
When possible, for every sequencing primer in a shipment, include a sequencing reaction with a known working combination of primer/DNA to be analyzed. As a benefit, if this is done several times for the same sequence, a confident consesus sequence for the sequenced region can be obtained.
After receiving sequence files, perform a BLAST search with the desired insert as query and the sequencing result as subject. If gaps/mutations are present, inspect the cromatogram file(s) to determine if the bases in question have been wrongly called.
If sequencing several candidates-clones putatively containing the same insert, perform a multiple alignment with the vector plasmid map/determined sequence as reference. Look for gaps/mutations that differ from the reference sequence and common to all the candidates, indicating a real mutation either during the cloning process or an error in the reference sequence/mutation in the original plasmid.
Biohacking Protocols: https://github.com/cathalgarvey/biohacking-protocols