Haynes Lab:Notebook/Engineering PC-TFs/2014/11/05: Difference between revisions
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==Summary== | ==Summary== | ||
* | * Gel Extracted/Purified BL01 | ||
* Retransformed BL01 & BL05 | |||
* Digested CMV/MV9 with XbaI for LCR | |||
* LCR | |||
* Digested CMV/MV9 with XbaI and desphos'd, clean and concentrated for long transformation | |||
* Long Transformation | |||
---- | |||
'''Gel Extraction/Purification of BL01'''<br> | |||
{| {{table}} border="1" cellspacing="3" | |||
<!-- Editing: the coding for this table is a bit more advanced. --> | |||
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<!-- The next two "rowspan=7" cells span all 7 rows in the table so that they can fit the "Expected" list and a gel image. rowspan is how you create merged rows in Wiki code. --> | |||
<!-- Replace Plasmid 1 and 2 with the names of your plasmids. Replace insert size and vector size with appropriate information for your plasmids. If you only have one Plasmid, delete all the text for Plasmid 2 | |||
<!-- After you upload your gel image to OWW, replace "GelImage.jpg" with the name of your image file --> | |||
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|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
| rowspan="7" | <u>Expected:</u><br>1. BL01 = 2500bp <br> | |||
|- | |||
| DNA(BL01) || 15.0 μL | |||
|- | |||
| 10X buffer || 3.0 μL | |||
|- | |||
| XbaI || 1.0 μL | |||
|- | |||
| SpeI || 1.0 μL | |||
|- | |||
| dH<sub>2</sub>O || 10 μL | |||
|- | |||
| Total || 30 μL --> 37°C/ 15 min. | |||
|} | |||
---- | |||
''' Gel Purification: BL01/V0120 ''' <br> | |||
* Using Prior Digest <br> | |||
* Placed 15μL 1kB Ladder in well one and 30μL of BL01 digest in well two. | |||
* Ran gel at 110V for 55 minutes. | |||
* Placed gel on UV transilluminator and cut out desired band(2500bp). | |||
* Used Zymo gel purification kit. | |||
* Assumed 200mg gel, added 600μL ADB to the 1.5mL tube containing extracted gel piece. | |||
* Put on heat block at 55°C for 8 minutes. | |||
* Put ~750μL gel/ADB into spin column, centrifuged at max rpm for 30 seconds. | |||
* Pipetted 200μL wash buffer to the tube, centrifuged at max rpm for 30 seconds. Repeated once. | |||
* Transferred column to new labeled 1.5mL tube. | |||
* Pipetted 15μL elution buffer to the column. | |||
* Spun at max rpm for 30 seconds. | |||
'''Concentration''' | |||
{| {{table}} | |||
|- bgcolor=#cfcfcf | |||
| Plasmid || OD260 || OD260/280 || ng/μL | |||
|- | |||
| 1. Purified BL01 || 0.001 || 2.831 || 10.694 | |||
|} | |||
<br> | |||
'''Digest and Dephos of CMV/MV9 with XbaI'''<br> | |||
{| {{table}} border="1" cellspacing="3" | |||
<!-- Editing: the coding for this table is a bit more advanced. --> | |||
<!-- valign="top" aligns all the text in the first row to the top. --> | |||
<!-- The | symbols on the next two lines start new cells in the same row. This does the same thing as ||, but you have to use | on new lines to set formatting for cells. --> | |||
<!-- bgcolor=#cfcfcf *colors* the *background* of the Reagent and Volume cells grey. --> | |||
<!-- The next two "rowspan=7" cells span all 7 rows in the table so that they can fit the "Expected" list and a gel image. rowspan is how you create merged rows in Wiki code. --> | |||
<!-- Replace Plasmid 1 and 2 with the names of your plasmids. Replace insert size and vector size with appropriate information for your plasmids. If you only have one Plasmid, delete all the text for Plasmid 2 | |||
<!-- After you upload your gel image to OWW, replace "GelImage.jpg" with the name of your image file --> | |||
<!-- is ASCII code for an invisible space. --> | |||
|- valign="top" | |||
|- | |||
| DNA(CMV/MV9) Concentration: 163ng/μL || 5.0 μL | |||
|- | |||
| 10X buffer || 1.5 μL | |||
|- | |||
| XbaI || 1.0 μL | |||
|- | |||
| dH<sub>2</sub>O || 7.5 μL | |||
|- | |||
| Total || 15 μL --> 37°C/ 15 min. | |||
|} | |||
<br> | |||
*Clean and concentrated XbaI cut CM9. Eluted with 10μL dH<sub>2</sub>O.<br> | |||
{| {{table}} border="1" cellspacing="3" | |||
<!-- Editing: the coding for this table is a bit more advanced. --> | |||
<!-- valign="top" aligns all the text in the first row to the top. --> | |||
<!-- The | symbols on the next two lines start new cells in the same row. This does the same thing as ||, but you have to use | on new lines to set formatting for cells. --> | |||
<!-- bgcolor=#cfcfcf *colors* the *background* of the Reagent and Volume cells grey. --> | |||
<!-- The next two "rowspan=7" cells span all 7 rows in the table so that they can fit the "Expected" list and a gel image. rowspan is how you create merged rows in Wiki code. --> | |||
<!-- Replace Plasmid 1 and 2 with the names of your plasmids. Replace insert size and vector size with appropriate information for your plasmids. If you only have one Plasmid, delete all the text for Plasmid 2 | |||
<!-- After you upload your gel image to OWW, replace "GelImage.jpg" with the name of your image file --> | |||
<!-- is ASCII code for an invisible space. --> | |||
|- valign="top" | |||
|- | |||
| Mixture from Above Digest || 10.0 μL | |||
|- | |||
| 10X Phosphotase buffer || 2.0 μL | |||
|- | |||
| Phosphotase || 1.0 μL | |||
|- | |||
| dH<sub>2</sub>O || 7.0 μL | |||
|- | |||
| Total || 20 μL --> 37°C/ 10 min. 75°C for 2 min. | |||
|} | |||
<br> | |||
*Clean and concentrated DNA after dephos step; eluted with 12μL dH<sub>2</sub>O.<br> | |||
{| {{table}} | |||
|- bgcolor=#cfcfcf | |||
| Plasmid || OD260 || OD260/280 || ng/μL | |||
|- | |||
| 1. Purified DNA || 0.059 || 2.046 || 59.108 | |||
|} | |||
'''LCR'''<br> | |||
LCR Calculations | |||
* DNA Part to Oligo Bridge is 1:10 (DNA part ratio is 1:1) | |||
* X ng insert = (bp inset / bp vector) x 1 x 50 ng vector | |||
* X ng BL01&9 = (2520bp/5197bp)*1*50ng vector = 24.24ng BL01&9 | |||
* Volume BL01&9 = 24.24ng*(1μL/10ng) = 2.424μL | |||
* Volume CMV/MV9 = 50ng*(1μL/33ng) = 1.28μL | |||
* 2:1 vector to insert use 2.56μL vector. | |||
{| class="wikitable" width=400px align="left" | |||
| || BL01|| BL05 | |||
|- | |||
| Insert DNA || 2.5μL || 2.5μL | |||
|- | |||
| XbaI Cut and Cleaned Vector DNA (CMV/MV9) || 1.3μL || 2.6μL | |||
|- | |||
| Oligo Bridge1 || 2.5μL || 2.5μL | |||
|- | |||
| Olido Bridge2 || 2.5μL || 2.5μL | |||
|- | |||
| 10X Ampligase Buffer || 2.5 μl || 2.5μL | |||
|- | |||
| Ampligase || 0.5μl || 0.5μL | |||
|- | |||
| Betaine || 0μL || 0μL | |||
|- | |||
| DMSO || 0μL || 0μL | |||
|- | |||
| dH<sub>2</sub>O || 13.2μL || 11.9μL | |||
|- | |||
| Total || 25.0 μL || 25μL | |||
|- | |||
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br> Placed in thermocycler on LCR setting. | |||
|} | |||
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'''Transformation''' | |||
<br> | |||
# Warmed selection agar plates at 37°C. | |||
# Incubated DH5α Turbo competent cells on ice just until thawed. Use 50 μL per transformation. | |||
# Added 50 μL thawed cells to 5μL LCR product. Immediately placed on ice and incubated for 10 min. (Do not heat shock; No 30 min. recovery is required for Amp resistance) | |||
# Pipetted the total volume of cells + LCR reaction onto the agar; spread using sterile glass beads. | |||
# Incubated overnight at 37°C. | |||
Revision as of 14:53, 5 November 2014
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Summary
Gel Extraction/Purification of BL01
Gel Purification: BL01/V0120
Concentration
LCR
Transformation
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