Sep 26, 2024
pGEM-T Cloning
- 1Washington University
Protocol Citation: Carolina Lopez 2024. pGEM-T Cloning. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvme389g3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 28, 2024
Last Modified: September 26, 2024
Protocol Integer ID: 100760
Abstract
Protocol for cloning in pGEM-T vector
Materials
Reagents:
- pGEM-T Easy Kit (Promega, A1360)
- Competent E. coli cells (NEB, C3040H)
- LB plates with necessary antibiotic
Before start
PRINCIPLES BEHIND THE PROCEDURE MUST BE UNDERSTOOD. PLEASE CONSULT WITH EXPERIENCED LAB MEMBER THE FIRST TIME YOU USE THIS PROCEDURE. UPDATE AS A GENERAL PROCEDURE AS NECESSARY BUT DO NOT MODIFY WITH SPECIFICS TO YOUR PROJECT, INSTEAD DOWNLOAD AND PASTE A MODIFIED COPY IN YOUR NOTEBOOK. Updated by MH, April 2023
Insert Generation
Insert Generation
1h 2m
1h 2m
1. Perform PCR to generate bands of interest in total volume of 50 µL .
2. Make 1% low melt-agarose gel.
a) Mix 1 g of Agar with 100 mL of TAE Buffer.
b) Microwave to boil agarose and let cool until you can touch bottle, but gel is not solid.
c) Add 1.5 µL of EtBr to agarose and pour into DNA gel mold with 10 well comb.
d) Let gel solidify.
3. Add 10 µL of loading buffer to PCR reaction then Load 60 µL of reaction into well of gel
4. Run gel for 00:45:00 at 120V.
5. Visualize band with UV light and cut out section of gel with band with new razor blade and place in 1.5 mL tube.
6. Purify Band from gel with Promega Wizard SV Gel and PCR Purification Kit (A9282)
b) Weigh DNA gel fragment and add 10 µL of Membrane Binding Solution per 10 mg of gel slice.
c) Incubate mixture at 65 °C for 00:10:00 or until gel is completely melted.
d) Add melted gel mixture to SV minicolumn in Collection Tube and incubate at room temperature for 00:01:00 .
e) Centrifuge at max speed for 00:01:00 . Discard flowthrough and reinsert column into tube.
f) Add 700 µL of Membrane Wash Solution. Centrifuge at max speed for 00:01:00 . Discard flowthrough and reinsert column into tube.
g) Add 500 µL of membrane wash solution. Centrifuge at max speed for 00:01:00 . Discard flowthrough and reinsert column into tube.
h) Spin empty column for 00:01:00 at max speed to remove excess ethanol.
i) Transfer column to labelled 1.5 mL tube and add 35 µL of NF H20. Incubate for 00:01:00 at room temperature.
j) Centrifuge at max speed for 00:01:00 .
k) Keep eluate and store at -20 °C .
1h 2m
pGEM Ligation Protocol
pGEM Ligation Protocol
2h 24m
2h 24m
1. Set up Ligation Reaction in 0.2 mL PCR tube
a) *Volume of PCR product should be 2 insert:1 pGEM vector molar ratio
b) For example:
| A | B | C | D | E | F | G | |
| Component | Length of DNA (bp) | Molar ratio | ng of DNA | Volume of 50ng/ul solution | |||
| pGEM-T Vector | 3015 | 1 | 50 | 1 ul | |||
| PCR Fragment | 500 | 2 | 16.58 | 0.33 ul | |||
| H20 | 2.67 ul |
2. Incubate for 01:00:00 at room temperature
3. Transform Product into E. coli
a) Add 2 µL of product to 50 µL of TOP10 cells and mix up and down slowly with a pipette.
b) Incubate for 00:20:00 on ice.
c) Heat shock bacteria in 42 °C waterbath for 00:01:00 .
d) Incubate on Ice for 00:03:00 .
e) Add 100 µL of SOC media and shake in warm room for 01:00:00 .
f) Plate bacteria onto LB-Antibiotic/Xgal Plate and incubate overnight at 37 °C
i. Add 40 µL of Xgal to plate and spread with plate spreader. Let dry
ii. Then plate 100 µL of bacteria onto plate.
iii. Spread cells with plate spreader to get individual colonies
g) Pick white colonies for miniprep growth and sequencing.
2h 24m
Protocol references