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== '''<span style="color:#558822"> Ligation </span>''' == | == '''<span style="color:#558822"> Ligation </span>''' == | ||
| - | + | Using NEB Quick Ligation kit: | |
1ul DNA ligase | 1ul DNA ligase | ||
| This page lists the protocols for all the experiments we conducted regularly. Unless otherwise noted, these are the standard procedures we used for each of the processes mentioned below. | 
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1.75mL of bacterial culture is added to 0.5ml of LB+50% glycerol.
1% w/v agarose gels were used in 1% TAE Buffer. Gels were run at 70v for 30mins.
Using the Phusion PCR kit:
25ul Phusion mix
1.25ul each primer (at 100uM)
2ul DNA
1.5ul DMSO
19ul water
1. 98C for 30s
2. 98C for 10s
3. 65C for 30s
4. 72C for 30s
5. 72C for 10min
6. 12C FOREVER
cycle 34x between 2-4
Using QIAprep Spin Miniprep kit:
1. Resuspend bacteria pellet in 250ul P1 buffer
2. Add 250ul P2 buffer and mix by inverting
3. Add 350ul N3 buffer and mix by inverting
4. Centrifuge 10mins at max, take supernatant and pipette into QIAprep spin column
5. Centrifuge 30-60s, discard flow through
6. Add 0.75ml buffer PE and centrigue 1min, discard flow through
7. Centrifuge an additional one min to remove residual buffer
8. Place QIAprep column in a new tube and add 50ul buffer EB, spin for 1min
35ul DNA
5ul Buffer (10x, NEB buffer 3)
0.5ul EcoRI
0.5ul PstI
0.5ul BSA (100x)
8.5ul water
Placed in 37C waterbath for 1hr
Using Zymoclean purification kit:
1. For each volume of agarose, add 3 volumes of ADB and incubate for 10mins at 55C.
2. Transfer melted agarose into a zymo-spin column and place in a collection tube, centrifuge at max for 1min and then discard liquid.
3. Add 200ul wash buffer and centrifuge at max for 30s
4. Add 10ul RNA-free water and spin for 1min.
Using NEB Quick Ligation kit:
1ul DNA ligase
10ul Buffer (2x)
Volume of insert and vector varies depending on concentration
Brought to 20ul total volume with water.
Leave at room temp for 5mins then put on ice.
1. Thaw cells (TOP10) on ice. To 25ul of cells add 5ul of the ligation and mix gently.
2. Incubate on ice for 30mins then heat shock at 42C for 30secs.
3. Place back on ice for 5mins before adding 1ml of pre-warmed SOC media.
4. Shake at 37C for 1hr at 225rpm.
5. Spread 100ul onto a pre-warmed selective plate and incubate overnight at 37C.
Amp is provided at 50mg/ml, we dilute to 50ug/ml in LB
Kan is provided at 30mg/ml, we dilute to 50ug/ml in LB
Chlor is provided at 34mg/ml, we dilute to 12.5ug/ml in LB.
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