Paul.xu: /* LR4 buffer (5x) - for use with attL1.1 and attL2.1 */

2014-05-07T03:23:21Z

LR4 buffer (5x) - for use with attL1.1 and attL2.1

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{| cellspacing="2" cellpadding="0" style="width: 862px; height: 488px;"
|-
| <br>
| <br>
|-
| Frame:
| style="font-family: Courier New;" | ACA AGT TTG TAC AAA AAA GCA GGC T------AC CCA GCT TTC TTG TAC AAA GTG GT
|-
| <br>
| style="font-family: Courier New;" | <br>
|-
|
Entry<br> clone

| style="font-family: Courier New;" | N75-ACAAGTTT<span style="color: rgb(0, 153, 0);">GTACAAAAAAGCAGGCT------------------ACCCAGCTTT</span>CTTGTACAAAGTGGT-N75<br>
N75-TGTTCAAACATGTTT<span style="color: rgb(0, 153, 0);" class="greenbackground">TTTCGTCCGA------------------TGGGTCGAAAGAACATG</span>TTTCACCA-N75<br> [-----------attL1-----------]        ORF       [-----------attL2-----------]

|-
|  
| style="font-family: Courier New;" | <div align="left">'''                                     X'''</div>
|-
| Destination<br>
vector

| style="font-family: Courier New;" | ACAAGTTT<span style="color: rgb(255, 102, 0);">GTACAAAAAAGCAGGCT-N100--------N100-ACCCAGCTTT</span>CTTGTACAAAGTGGT<br>
TGTTCAAACATGTTT<span style="color: rgb(255, 102, 0);">TTTCGTCCGA-N100--------N100-TGGGTCGAAAGAACATG</span>TTTCACCA<br> [-----------attR1------------]  ccdB  [-----------attR2------------]

|-
|  
| style="font-family: Courier New;" |
'''                                     ='''

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| Expression<br>
clone

| style="font-family: Courier New;" |
ACAAGTTT<span style="color: rgb(0, 153, 0);">GTACAAAAAAGCAGGCT------------------ACCCAGCTTT</span>CTTGTACAAAGTGGT<br>

TGTTCAAACATG<span style="color: rgb(0, 153, 0);">TTT</span><span style="color: rgb(0, 153, 0);">TTTCGTCCGA------------------TGGGTCGAAAGAACATG</span>TTTCACCA<br> [--------attB1----------]        ORF       [--------attB2----------]

<br>

|-
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| style="font-family: Courier New;" | <div align="left">'''                                     +'''</div>
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By-<br> product

| style="font-family: Courier New;" | N75-ACAAGTTT<span style="color: rgb(255, 102, 0);">GTACAAAAAAGCAGGCT-N100--------N100-ACCCAGCTTT</span>CTTGTACAAAGTGGT-N75<br>
N75-TGTTCAAACATG<span style="color: rgb(204, 102, 0);">TTT</span><span style="color: rgb(204, 102, 0);">TTTCGTCCGA-N100--------N100-TGGGTCGAAAGAACATG</span>TTTCACCA-N75<br> [-------------attP1--------------]  ccdB  [-------------attP2--------------]

|}

<br> LR reaction

#Assemble the reaction:

{| width="75%"
|-
|  
|  
| <div align="right">5 µl</div>
| <div align="right">10 µl</div>
|-
|  
| Entry clone (50-150ng)
| <div align="right">x µl</div>
| <div align="right">x µl</div>
|-
|  
| Destination vector
| <div align="right">75 ng</div>
| <div align="right">150 ng</div>
|-
|  
| 5X LR Buffer
| <div align="right">1 µl</div>
| <div align="right">2 µl</div>
|-
|  
| TE pH 8.0
| <div align="right">to 4 µl</div>
| <div align="right">to 8 µl</div>
|-
| width="7%" |  
| width="64%" | LR Clonase
| width="15%" | <div align="right">0.5µl</div>
| width="15%" | <div align="right">1 µl</div>
|}

#Incubate at 25°C for 1 hour to overnight.
#Transform into competent cells and plate on plates containing a suitable antibiotic.

<br>

== original BP buffer (5x) ==

*125 mM Tris HCl pH 7.5
*25 mM EDTA
*110 mM NaCl
*25 mM Spermidine-HCl
*5 mg/ml BSA

Note: BSA should be NON-acetylated, as Int is rapidly inactivated (at least in its pure form) by acetylated BSA

== BP3 buffer (5x) - only works with ''att''B1.1 and ''att''B2.1 ==

*100 mM Tris pH 7.5
*20 mM EDTA
*225 mM NaCl
*30 mM Spermidine-HCl
*25% Glycerol

== Original LR buffer (5x) ==

*250 mM of Tris HCl pH 7.5
*2.25 mM of EDTA
*250 mM of NaCl
*12.5 mM of spermidine HCl
*1 mg/mL of BSA

Note: BSA should be NON-acetylated, as Int is rapidly inactivated (at least in its pure form) by acetylated BSA

== LR4 buffer (5x) - for use with ''att''L1.1 and ''att''L2.1 ==

*200 mM Tris-Cl, pH 7.5
*10 mM EDTA
*320 mM NaCl
*35 mM spermidine-HCl
*25% Glycerol

Plasmids:<br>

pMCSG13-IHF, Clonesdev 405<br>

pET28b-Int, 406<br>

pET28b-Xis, 407<br>

Patents
5,888,732, 6,143,557, 6,171,861, 6,270, 969, and 6,277,608

[[Category:Protocols]]

Gateway Enzymes - Revision history

Paul.xu: /* LR4 buffer (5x) - for use with attL1.1 and attL2.1 */