Restriction Digest 8-3

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Current revision (12:06, 4 August 2009) (view source)
 
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The first 3 lanes are digestons from the first gel that I had some left over. The last 2 lanes are constructs 13 and 17 redone. Under the Treefort, I could see construct 17 very, very faintly. I continued to see nothing for 13. I then proceeded to do a colony PCR for 13 and got nothing. The gel is blank so it isn't interesting.
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The first 3 lanes are digestons from the first gel that I had some left over. The last 2 lanes are constructs 13 and 17 redone. For 11, I couldn't confirm the second band at 600 this time. It was probably due to contamination from 10 right next to it which is at 600 bp. Under the Treefort, I could see construct 17 very, very faintly. I continued to see nothing for 13. I then proceeded to do a colony PCR for 13 and got nothing. The gel is blank so it isn't interesting.
[[Image:Upload3.jpg]]
[[Image:Upload3.jpg]]

Current revision

Below is a mapping of the 21 constructs. All of the constructs except for 13 (C09ig0033 {<CPG_L6>}(dblterm)) check out. The constructs go from left to right in the below order:

#     ID        Construct                       Length   
1     C09ig0027	{<VtaA11 AtD>}(dblterm)	        378
2     C09ig0026	{<cl02365 AtD>}(dblterm)	963
3     C09ig0035	{<ehaB>}(dblterm)	        1365
4     C09ig0028	{<Hag AtD>}(dblterm)	        378
5     C09ig0024	{<azo1653 AtD>}(dblterm)	969
6     C09ig0029	{<Pcryo_1225 AtD>}(dblterm)	381
7     C09ig0025	{<OprF AtD>}(dblterm)	        819
8     C09ig0030	{<Hia AtD>}(dblterm)	        378
9     C09ig0031	{<upaG_short>}(dblterm)	        405
10    C09ig0036	{<eCPX>}(dblterm)	        612
11    C09ig0032	{<CPG_L2>}(dblterm)	        999
12    C09ig0037	{<TshA>}(dblterm)	        1710
13    C09ig0033	{<CPG_L6>}(dblterm)	        999
14    C09ig0039	{<yuaQ AtD>}(dblterm)	        1008
15    C09ig0034	{<espP(beta)>}(dblterm)	        1488
16    C09ig0040	{<AIDA-1 AtD>}(dblterm)         1560
17    C09ig0019	{<Cub>}(dblterm)	        273
18    C09ig0023	{<Tev C>}(dblterm)	        513
19    C09ig0020	{<Nub>}(dblterm)	        282
20    C09ig0021	{<caspace 3>}(dblterm)   	969
21    C09ig0022	{<Tev N>}(dblterm)	        495

The gel on the left is slightly bent which is why all of the constructs are ~100bp above what they should be. The two missing were 13 and 17. All of the constructs minus 13 and 17 could be seen under the Treefort. A lot couldn't be seen from this picture. There also seemed to be a second faint band for 11 (other than the correct band on this picture) which can be seen from the treefort. The second band is just above the 500 bp ladder band.

Image:Upload.jpg

I ran another gel on just 11, 13 and 17. on the second gel, from left to right, the ordering goes:

#     ID        Construct                       Length   
11    C09ig0032	{<CPG_L2>}(dblterm)	        999         From first digestion
13    C09ig0033	{<CPG_L6>}(dblterm)	        999         From first digestion
17    C09ig0019	{<Cub>}(dblterm)	        273         From first digestion
13    C09ig0033	{<CPG_L6>}(dblterm)	        999         New digestion
17    C09ig0019	{<Cub>}(dblterm)	        273         New digestion

The first 3 lanes are digestons from the first gel that I had some left over. The last 2 lanes are constructs 13 and 17 redone. For 11, I couldn't confirm the second band at 600 this time. It was probably due to contamination from 10 right next to it which is at 600 bp. Under the Treefort, I could see construct 17 very, very faintly. I continued to see nothing for 13. I then proceeded to do a colony PCR for 13 and got nothing. The gel is blank so it isn't interesting.

Image:Upload3.jpg

Just to confirm things. I also ran a Eco/XhoI digest on 13 and 17. 13 and 17 are the far right lanes. With 17 being the far right and 13 being second to far right. As you can see, 17 cuts with Eco/XhoI properly, but 13 fails to cut despite how bright the vector band is.

Image:Upload2.jpg

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