Table 1. Second-strand synthesis with limiting primer amounts

DNA ng)	T7 primer (μL)^a	NEB Buffer 2 (μL)	5 mM dNTPs (μL)	H₂O (μL)	Tailed DNA (μL)	Klenow (μL)	Total volume (μL)
>75	0.60 (25 μM)	5.0	2.0	20.4	20.0	2.0	50
50-75	0.30 (25 μM)	2.5	1.0	0.20	20.0	1.0	25
25	0.15 (25 μM)	2.5	1.0	0.35	20.0	1.0	25
10^b	1.50 (1 μM)	1.0	0.4	0.20	6.5^b	0.4	10
5^b	0.75 (1 μM)	1.0	0.4	0.95	6.5^b	0.4	10
2.5^b	0.38 (1 μM)	1.0	0.4	1.32	6.5^b	0.4	10
^aUsing limiting amounts of primer is highly advisable when amplifying from very small amounts of starting material. Not only will this decrease the amount of primer-dimer product (see the Troubleshooting section of Whole Genome Amplification by T7-based Linear Amplification of DNA (TLAD): III. Sample Purification [Liu et al. 2008c]), but it may also increase the yield of the desired amplification product. The table gives the single reaction volumes to use for a suggested mass range of starting material.
^bThe tailed DNA will need to be dried down in a vacuum centrifuge to the volume indicated for reaction volumes that total 10 μL.