User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/04/22: Difference between revisions
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''' | '''Building a Reporter Gene to Determine Chromatin Protein Function (Ligation into the mammalian vector)''' | ||
# | |||
*Ligation of construct KAH182-KAH201 and mammalian vector V0200 and cloning in DH5α-T. | |||
*KAH182 = BBa_S04739 and KAH201 = BBa_S04745 and V0200 = BBa_J176121. | |||
*Assembly strategy: The backbone V0200(N/S)/4442 , the insert is KAH182-KAH201(N/S)/1901. | |||
'''Digests''' <br> | |||
[[Image:KAH182-KAH201-V0200-S-N Digestion.jpg|thumb|350px| (1) KAH182-KAH201, 1901 bp, (2) V0200, 4442 bp, Both 1 and 2 are cut with Spe1 and Not11]] | |||
{| {{table}} cellspacing="3" width=350px | |||
| Plasmid DNA || 25.00 μl | |||
|- | |||
| NotI || 1.0 μl | |||
|- | |||
| SpeI || 1.0 μl | |||
|- | |||
| 10x FastDigest buffer + green loading dye || 3.00 μl | |||
|- | |||
| dH<sub>2</sub>O || 0.00 μl | |||
|- | |||
| || 30.0 μl total | |||
|- | |||
| Incubate at 37°C for 10 minutes. | |||
|} | |||
* '''Ligations''' | |||
{| {{table}} cellspacing="3" <!-- Ligations table --> | |||
|- bgcolor=#cfcfcf | |||
| Ligation || <font color="blue"><u> | |||
|- | |||
| 1. E. coli DH5α-T KAH182-KAH201(N/S)/size, 9.00 ng + V0200(N/S)/size, 48.00ng || <font color="blue">KAH182/KAH201 3:1 No Colonies</font> | |||
|- | |||
| 2. E. coli DH5α-T KAH182-KAH201(N/S)/size, 9.00 ng + V0200(N/S)/size, 48.00ng || <font color="blue">KAH182/KAH201 4:1 One Colony</font> | |||
|- | |||
| 3. E. coli DH5α-T KAH182-KAH201(N/S)/size, 9.00 ng + V0200(N/S)/size, 48.00ng || <font color="blue">KAH182/KAH201 4:1 one Colony</font> | |||
|- | |||
| 4. KAH201(S/P)/size, 36.29ng (Control Plate)|| <font color="blue">One Colonies and lots of satellite colonies in the middle of the pate</font> | |||
|} | |||
* Calculations are for the ng of insert we need to get a 3:1, 4:1 and 5:1 ratios of insert molecules to 20 ng vector molecules | |||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | |||
| || 1 (3:1) || 2 (4:1) || 3 (5:1) || 4 (- Ctrl) | |||
|- | |||
| Insert DNA || 2.85 || 3.80 || 4.76 || --- | |||
|- | |||
| Vector DNA || 1.00 || 1.00 || 1.00 || 1.00 | |||
|- | |||
| 2x lgn buf (Roche) || 5.00 || 5.80 || 6.76 || 5.0 | |||
|- | |||
| T4 ligase (NEB) || 1.0 || 1.0 || 1.0 || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 0.15 || 0.00 || 0.00 || 3.00 | |||
|- | |||
| || 10.00 μL || 11.60 μL || 13.52μl || 10μL | |||
|} | |||
* The incubation time for the ligation process was 15min at room temperature. | |||
* Fast transformation, 15 min on ice. |
Revision as of 13:19, 23 April 2013
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DateBuilding a Reporter Gene to Determine Chromatin Protein Function (Ligation into the mammalian vector)
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