Editing TALEN Real Assembly (section)

== Construction of TAL Arrays  ==

9,&nbsp; Using the graphical output generated by ZiFiT Targeter as a guide,&nbsp;perform the series of ligations indicated in the first row of the&nbsp;REAL or REAL-Fast figure by repeating steps 10 - 19 below for each ligation. The numbers above each&nbsp;TALE repeat unit or array in the graphical guide identify the names of the&nbsp;plasmids that are to be used for each&nbsp;ligation. For each pair to be ligated, the TALE repeat on the left is&nbsp;the amino-terminal repeat and the one on the right is the carboxy-&nbsp;terminal repeat. For each ligation, a DNA vector backbone encoding the&nbsp;amino-terminal repeat(s) is ligated to a fragment encoding the carboxy-&nbsp;terminal repeat(s).&nbsp; 

10,&nbsp; Digest plasmid(s) encoding the amino-terminal TALE repeat(s) with&nbsp;BamHI and BsaI using the following conditions: 

Plasmid encoding amino-terminal TALE repeat <strike>1.5 µg&nbsp;</strike> 300 ng<strike><br>10X Buffer (NEBuffer 4) 4 µl&nbsp;</strike>&nbsp; 10 X FastDigest Buffer: 1.5 ul<br><strike>Bovine Serum Albumin (10mg/ml) 4 µl&nbsp;</strike><br><strike>BsaI (5 U/µl) 2 µl or&nbsp;</strike>&nbsp;&nbsp; FastDigest BbsI (BpiI), 0.5 ul<br><strike>BamHI (20 U/µl) 2 µl&nbsp;</strike> FastDigest BamH1, 0.5 ul<br> 

Add nuclease-free water to a total volume of <strike>40 µl , </strike>15 ul<br>Incubate the reaction at 37°C for <strike>2 hours</strike>&nbsp; 30 min. 

11,&nbsp; Isolate the vector backbone from the restriction digest of step&nbsp;<strike>10&nbsp;on a 5% non-denaturing polyacrylamide gel</strike> &nbsp; 1.5% agaose gel and purify the DNA (add 0.7vol of isopropanol) <strike>according&nbsp;to the protocol found in Box 2 of Maeder et al., Nat Protocols 2009</strike>. <strike>Resuspend the final DNA pellet in 20 µl of nuclease-free water.&nbsp;&nbsp; </strike>Elute with 20 ul H2O<br> 

<br>12,&nbsp; Digest plasmid(s) encoding the carboxy-terminal TALE repeat(s)&nbsp;with BbsI and BamHI using the following conditions:&nbsp; 

Plasmid encoding carboxy-terminal TALE repeat(s) <strike>1.5 µg&nbsp;&nbsp;&nbsp; </strike>300 ng<br><strike>NEB Buffer 2 4 µl&nbsp;</strike>&nbsp; 10 X FastDigest Buffer: 1.5 ul<br><strike>Bovine Serum Albumin (10mg/ml) 4 µl&nbsp;</strike><br><strike>BbsI (5 U/µl) 2 µl</strike>&nbsp;&nbsp; FastDigest BbsI (BpiI): 0.5 ul<br><strike>BamHI (20 U/µl) 2 µl</strike>&nbsp; FastDigest BamH1: 0.5 ul 

Add nuclease-free water to a total volume of <strike>40 µl </strike>&nbsp; 15 ul<br>Incubate the reaction at 37°C for <strike>2 hours</strike>&nbsp; 30 min 

13,&nbsp; Isolate the DNA fragment encoding the TALE repeat(s) from the&nbsp;restriction digest of step 12 on <strike>a 5% non-denaturing polyacrylamide&nbsp;gel and purify the DNA using the protocol found in Box 2 of Maeder et&nbsp;al., Nat Protocols 2009.</strike> <strike>Resuspended the final DNA pellet in 20 µl of nuclease-&nbsp;free water.</strike> <br> 

1.5% agaose gel and purify the DNA (add 0.7vol of isopropanol), Elute with 20 ul H2O<br> 

Note that the size of the DNA fragment will be equal to N&nbsp;x ~102 bps where N is the number of TALE repeats encoded on the&nbsp;fragment.&nbsp; 

14,&nbsp; Ligate the purified DNA fragment isolated in step 13 into the&nbsp;purified vector backbone isolated in step 11 as follows: 

Purified vector backbone (from step 11) <strike>1 µl&nbsp;&nbsp; </strike>5 ul<br>Purified fragment (from step 13) or water <strike>3 µl </strike>&nbsp; 5 ul<br><strike>Quick Ligase Buffer (NEB) 4.5 µl&nbsp;<br>T4 DNA Ligase (400U/ul) 0.5 µl&nbsp;</strike><br> 

T7 ligase, 0.5 ul<br> 

2X T7 ligase buffer, 10 ul<br> 

<br>Total <strike>9</strike> 20 µl&nbsp;<br>Incubate this reaction at room temperature for <strike>15</strike> 30 minutes.<br>Also perform a control ligation using vector backbone alone (i.e.--without&nbsp;fragment). 

15, Transform each ligation from step 14 into 100 <strike>90</strike> µl chemically&nbsp;competent <strike>XL1-Blue</strike> MACH cells as follows:<br> 

<br>Mix the ligations with competent cells and&nbsp;incubate on ice for 5 minutes. Perform heat shock at 42°C for 1 minute and return the transformations to ice for 1 minute. Add 500 µl LB to the cells and recover&nbsp;with agitation for 45 minutes at 37°C. <br> 

Spin down cell and plate <strike>200 µl of</strike> each&nbsp;transformation on an LB plate supplemented with 100 µg ml-1&nbsp;carbenicillin. Grow the transformed cells at 37°C for 12 – 14 hours.&nbsp; 

16,&nbsp; Ensure that the actual ligation/transformation of step 15 yields at least&nbsp;10-fold more colonies than the control ligation (backbone only) and then inoculate two single&nbsp;colonies from the actual ligation/transformation plate into 4 ml of&nbsp;LB&nbsp;supplemented with carbenicillin 100 mg ml–1. Allow these cells to grow overnight at 37°C with agitation. 

CRITICAL STEP: To reduce the risk of plasmid deletions, do not allow&nbsp;the cultures to grow for more than 14 hours.&nbsp; 

17,&nbsp; Use a <strike>QIAprep</strike> spin Miniprep kit to isolate plasmid DNA from these overnight cultures.&nbsp; 

18,&nbsp; Digest candidate plasmids with XbaI and BamHI to determine whether they have successfully taken up the fragment encoding the carboxy-terminal TALE&nbsp;repeat(s) as follows: 

Plasmid <strike>1 µg </strike>&nbsp; 200 ng<br><strike>10X Buffer (NEB Buffer 4) 4 µl </strike>&nbsp; FastDigest 1.5 ul<br><strike>Bovine Serum Albumin (10mg/ml) 4 µl&nbsp;</strike><br><strike>XbaI (20 U/µl) 1 µl </strike>&nbsp; FastDigest Xba1, 0.5 ul<br><strike>BamHI-HF (20 U/µl) 1 µl</strike>&nbsp; FastDigest BamHI 0.5 ul<strike><br></strike> 

Add nuclease-free water to a total volume of <strike>40 µl&nbsp; </strike>15 ul<br>Incubate at 37°C for <strike>an hour.</strike>&nbsp; 30 min 

19, Visualize the DNA fragments generated by the restriction digests from step 18 on&nbsp;a 5% non-denaturing polyacrylamide gel. Plasmids that have&nbsp;successfully taken up the fragment encoding the carboxy-terminal TALE&nbsp;repeat(s) should yield a ([(M + N) x 102] + 33) bp fragment (where M&nbsp;and N are the numbers of TALE repeats encoded by the vector backbone&nbsp;and fragment, respectively.) 

20, Following completion of the first set of ligations, perform the&nbsp;series of ligations indicated in the second row of the graphical&nbsp;guide&nbsp;by using steps 10 - 19 above.&nbsp; 

21, Continue performing ligations in subsequent rows of the graphical&nbsp;guide using steps 10 - 19 above until the final assembled array is&nbsp;completed.&nbsp; 

Cloning TAL arrays into the nuclease backbone 

22, Digest the specific TALEN expression vector indicated at the&nbsp;bottom of the graphical output (pJDS70, pJDS71, pJDS74 or pJDS78; available from Addgene)&nbsp;with&nbsp;BsmBI restriction enzyme as follows. 

TALEN Expression Vector <strike>2µg&nbsp;</strike>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 200 ng<br><strike>10x Buffer (NEB Buffer #3) 5 µl</strike>&nbsp;&nbsp; FastDigest Buffer, 1.5 ul<br><strike>BsmBI (10U/ul) 5 µl</strike>&nbsp; FastDigest BsmBI (Esp3I) 0.5 ul<br> 

Add nuclease-free water to a total volume of <strike>50</strike> 15 µl&nbsp;<br>Incubate at 55°C for <strike>3 hours</strike>&nbsp; 30 min. 

23, Isolate the vector backbone on a <strike>5% non-denaturing polyacrylamide gel</strike> agarose gel and purify the DNA with spin column and elute with 20 ul water. <strike>using&nbsp;<br>the&nbsp;protocol found in Box 2 of Maeder et al., Nat Protocols 2009</strike>. <strike>Resuspend the final pellet in 20 µl of nuclease-free water.&nbsp;&nbsp;</strike> 

24, Digest plasmid(s) encoding the final assembled TALE repeat array&nbsp;(from step 21 above) with BbsI and BsaI as follows: 

Plasmid encoding assembled TALE repeat array <strike>1.5 µg&nbsp; </strike>200 ng<br><strike>10X Buffer (NEB Buffer 2) 4 µl&nbsp;<br>Bovine Serum Albumin (10mg/ml) 4 µl&nbsp;</strike><br>Fast Digest BbsI <strike>(5 U/µl)</strike> <strike>2</strike> 0.5 µl&nbsp;<br>Fast Digest BsaI <strike>(10 U/µl)</strike> <strike>2</strike> 0.5 µl 

Add nuclease-free water to a total volume of <strike>40</strike> 15 µl&nbsp;<br>Incubate at 37°C for <strike>2 hours</strike>.30 min.<br> 

25, Isolate the DNA fragment encoding the TALE repeat array(s) from&nbsp;the restriction digest of step 24 on an agarose gel a <strike>5% non-denaturing&nbsp;polyacrylamide gel and purify the DNA using the protocol found in Box&nbsp;2 of Maeder et al., Nat Protocols 2009</strike>.&nbsp; <br> 

<strike>Resuspend the final DNA pellet in 20&nbsp;µl of nuclease-free water.</strike> <br> 

Purify with spin column and elute with 20 ul water.<br> 

Note that the size of the DNA fragment&nbsp;will be equal to N x ~102 bps where N is the number of TALE repeats&nbsp;encoded on the fragment.&nbsp; 

26, Ligate the purified DNA fragment isolated in step 25 into the&nbsp;purified TALEN expression vector backbone isolated in step 23 as&nbsp;follows: &nbsp; 

<strike>Purified vector backbone (from step 23) 1 µl&nbsp;<br>Purified fragment (from step 25) or water 3 µl&nbsp;<br>Quick Ligase Buffer (NEB) 4.5 µl&nbsp;<br>T4 DNA Ligase (400U/ul) 0.5 µl&nbsp;<br>Total 9 µl&nbsp;</strike><br> 

Purified vector backbone (from step 23) &nbsp; 5 ul<br>Purified fragment (from step 25) or water&nbsp; 5 ul<br>T7 ligase, 0.5 ul 

2X T7 ligase buffer, 10 ul 

<br>Total&nbsp; 20 µl <br> 

<br>Incubate at room temperature for&nbsp;&nbsp; <strike>15&nbsp;</strike>&nbsp; 30 minutes. <br>Also perform a vector backbone only ligation as a control. 

27, Transform each ligation from step 26 into 90 µl chemically&nbsp;competent<strike>XL1-Blue</strike> MACH cells as follows:<br>Mix the ligations with competent cells and&nbsp;incubate on ice for 5 minutes. Perform heat shock at 42°C for 1 minute and return the transformations to ice for 1 minute. Add 500 µl LB to the cells and recover&nbsp;with agitation for 45 minutes at 37°C. Plate 200 µl of each&nbsp;transformation on an LB plate supplemented with 100 µg ml-1&nbsp;carbenicillin. Grow the transformed cells at 37°C for 12 – 14 hours.&nbsp; 

28, Ensure that the actual ligation/transformation of step 15 yields at least&nbsp;10-fold more colonies than the control ligation (backbone only) and then inoculate two single&nbsp;colonies from the actual ligation/transformation plate into 4 ml of&nbsp;LB&nbsp;supplemented with carbenicillin 100 mg ml–1. Allow these cells to grow overnight at 37°C with agitation.&nbsp; 

CRITICAL STEP: To reduce the risk of plasmid deletions, do not allow&nbsp;the overnight bacterial cultures to grow for more than 14 hours.&nbsp; 

29, Use a QIAprep spin Miniprep kit to isolate plasmid DNA from these overnight cultures.<br>30, Digest the candidate plasmids with FastDigest KpnI<strike>-HF </strike>and BamHI<strike>-HF</strike> to determine whether they have successfully taken up the fragment encoding the carboxy-terminal TALE&nbsp;repeat(s) as follows: 

Plasmid <strike>0.5 </strike>µg 200 ng<br><strike>10X Buffer (NEBuffer 4) 5 µl&nbsp;<br>Bovine Serum Albumin (10 mg/ml) 5 µl&nbsp;</strike><br> 

10 X FastDigest Buffer<br>FastDigest KpnI<strike>-HF (20 U/µl) 1</strike> 0.5 µl&nbsp;<br>FastDigest BamHI<strike>-HF (20 U/µl) 1</strike> &nbsp;0.5 µl&nbsp; 

Add nuclease-free water to a total volume of <strike>50&nbsp;</strike>&nbsp; 15 µl&nbsp;<br>Incubate for <strike>2 hours</strike> 30 min at 37 °C 

31, Visualize the fragment generated by the restriction digests from step 30 on&nbsp;a <strike>5% non-denaturing polyacrylamide gel</strike> agarose gel. Plasmids that have&nbsp;successfully taken up the fragment encoding the TALE repeat array&nbsp;should yield a (650 + [N x ~102]) bp fragment (where N is the number&nbsp;of TALE repeats encoded in the array.) 

32, Optional: TALEN expression plasmids can be sequence-verified by&nbsp;DNA sequencing using forward primer JDS2978 and reverse primer&nbsp;JDS2980. <br>33. The nucleotide sequence of the final product is provided at the bottom of the graphical guide generated by ZiFiT Targeter.<br> 

[[Category:Protocols]] [[Category:GenomeEngineering]]