1.Wet lab: colony PCR screen
When it fits: Use this when you are a graduate student screening clones, half the colonies will be empty vector, and in a month you need to know exactly which tube came from which colony.
JH-2026-112: Colony PCR screen of pJH14 transformants, 2026-10-20
Project: [[mCherry reporter construct]] Author: Ji-woo Han Reviewer or witness: Dr. Okonjo, initialed 2026-10-22 Continues from: [[JH-2026-111 Ligation and transformation]]
Objective
Find colonies carrying the 750 bp mCherry-NLS insert in the pJH14 backbone.
Expected result
With vector-flanking primers VF/VR, insert-positive colonies give about 1,060 bp; empty vector gives 310 bp (sizes from [[pJH14 plasmid map]]).
Materials and equipment
- 2x Taq master mix, lot 24C-0918, freezer box B3
- Primers VF and VR, 10 µM working stocks made 2026-09-30
- LB-kanamycin plate from JH-2026-111, 23 colonies after overnight at 37 °C
- Thermocycler 2 (bay 4), gel box A, 1 kb ladder lot 0731
Procedure
Protocol: [[Colony PCR v3]]
- Picked 8 colonies (C1 to C8) with sterile tips, touched each to a numbered patch plate, then swirled into 25 µL reactions: 12.5 µL master mix, 1 µL VF, 1 µL VR, 10.5 µL water.
- Negative control: no colony (NTC). Positive control: 10 ng pJH9 (known insert).
- Cycling: 95 °C 5 min; 30 cycles of 95 °C 30 s, 55 °C 30 s, 72 °C 75 s; 72 °C 5 min.
- 5 µL of each on a 1% agarose gel, 100 V, 40 min.
Deviations from protocol: Annealing at 55 °C instead of 58 °C. Thermocycler 1 was booked and block 2 runs about 2 °C hot by the last calibration, so I set it lower.
Observations and raw data
- 14:10 Patch plate labeled P-1 to P-8, at 37 °C.
- 16:45 Gel imaged. Bands near 1,050 bp in C2, C5, C7. Bands near 300 bp in C1, C3, C4, C8. No band in C6. NTC clean. Positive control at expected size.
- Data files: GelDoc, /HanJ/2026-10/JH112_colonyPCR.tif
Results
3 of 8 colonies (C2, C5, C7) carry an insert of the right size. C6 failed to amplify, likely too little template.
Conclusion and next steps
- 37.5% positive, in line with the last two ligations at this vector:insert ratio.
- Miniprep C2 and C5 from patch plate, send for sequencing with VF @due(2026-10-21)
- Keep C7 patch at 4 °C as backup @due(2026-10-21)
Corrections
- 2026-10-21, JH: first line of Observations said "P-1 to P-9." There were 8 patches. Typo, not a ninth colony.